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Trichoderma longibrachiatum UN32 is an industrially important fungus capable of producing Dendrobine-Type Total alkaloids (DTTAs). Several reports have pointed out that branched-chain amino acid aminotransferases (BCATs) participate in backbone modification or promote the production of secondary metabolites. We previously proposed that cobalt chloride increased DTTAs production in T. longibrachiatum UN32, which was associated with enhanced expression of the gene BCAT, TlBCAT (Genbank accession No. PP465542). Following cloning and characterization, the cDNA of TlBCAT was found to consists of 1191 bp, encoding a protein of 397 amino acid residues. The molecular mass of TlBCAT was about 42 kDa through SDS-PAGE analysis. The predicted pI value was 5.54. Recombinant TlBCAT can catalyze L-leu with a catalytic efficiency of 15.91 mM - 1 S - 1 . In the binding pocket, residues interacting with PLP, including Tyr68, Arg93, Tyr136, and Lys194, are highly conserved. TlBCAT exhibits a broad spectrum of substrate specificity, typical for catalyzing the transamination reaction of various branched-chain and hydrophobic amino acids, with α-ketoglutarate as the amino acceptor. Exogenous branched-chain amino acids (BCAAs) positively affect Trichoderma growth and DTTAs production. These findings offer insights into the physiological significance of BCAAs and present a novel approach for enhancing DTTAs production in Trichoderma mycelium cultures., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Environmental pollution is growing every day in terms of the increase in population, industrialization, and urbanization. Shewanella azerbaijanica is introduced as a highly potent bacterium in metal bioremediation. The mtrC gene was selected as a cloning target to improve electron flux chains in the EET (extracellular electron transfer) pathway. Using the SDM (site-directed mutagenesis) technique, the unique gene assembly featured the mtrC gene sandwiched between two napD/B genes to disrupt the nitrate reduction pathway, which serves as the primary metal reduction competitor. Shew-mtrC gene construction was transferred to expression plasmid pET28a (+) in the expression host bacteria (E. coli BL21 and S. azerbaijanica), in pUC57, cloning plasmid, which was transferred to the cloning host bacteria E. coli Top10 and S. azerbaijanica. All cloning procedures (i.e., synthesis, insertion, transformation, cloning, and protein expression) were verified and confirmed by precise tests. ATR-FTIR analysis, CD, western blotting, affinity chromatography, SDS-PAGE, and other techniques were used to confirm the expression and structure of the MtrC protein. The genome sequence and primers were designed according to the submitted Shewanella oneidensis MR-1 genome, the most similar bacteria to this native species. The performance of recombinant S. azerbaijanica bacterium in metal bioremediation, as sustainable strategy, has to be verified by more research., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Interleukin (IL) 36 is a member of the IL-1-like proinflammatory cytokine family that has a protective role in mucosal immunity. We hypothesized that mucosal delivery of IL-36γ to the intestine would be a very effective way to prevent intestinal diseases. Here, we genetically engineered a lactic acid bacterium, Lactococcus lactis, to produce recombinant mouse IL-36γ (rmIL-36γ). Western blotting and enzyme-linked immunosorbent assay results showed that the engineered strain (NZ-IL36γ) produced and hypersecreted the designed rmIL-36γ in the presence of nisin, which induces the expression of the recombinant gene. We administered NZ-IL36γ to mice via oral gavage, and collected the ruminal contents and rectal tissues. Colony PCR using primers specific for NZ-IL36γ, and enzyme-linked immunosorbent assay to measure the rmIL-36γ concentrations of the ruminal contents showed that NZ-IL36γ colonized the mouse intestines and secreted rmIL-36γ. A microbiota analysis revealed increased abundances of bacteria of the genera Acetatifactor, Eubacterium, Monoglobus, and Roseburia in the mouse intestines. Real-time quantitative PCR of the whole colon showed increased Muc2 expression. An in vitro assay using murine colorectal epithelial cells and human colonic cells showed that purified rmIL-36γ promoted Muc2 gene expression. Taken together, these data suggest that NZ-IL36γ may be an effective and attractive tool for delivering rmIL-36γ to improve the intestinal environment., (© 2024. The Author(s).)

Alkyl hydroperoxide reductase subunit C (AhpC) contributes to the cellular defense against reactive oxygen species. However, it remains understudied in psychrophiles. Amino acid comparison demonstrated that AhpC from Psychrobacter sp. ANT206 (ANT206) (PsAhpC) revealed fewer numbers of Lys and more numbers of Gly, which might have favored higher flexibility at low temperature. The recombinant PsAhpC (rPsAhpC) was most active at 25 °C and retained 35% of its residual activity at 0 °C, indicating that it was a cold-adapted enzyme. Additionally, rPsAhpC demonstrated significant salt tolerance, sustaining its activity in the presence of 4.0 M NaCl. Molecular dynamics simulations indicated that PsAhpC had comparatively loose conformation, which facilitated reactions at low temperatures. Subsequently, an ahpc knockout mutant was constructed, and the growth rate of the knockout mutant significantly decreased, suggesting that ahpc might be crucial for the growth of ANT206 at low temperatures. The findings provide a robust foundation for further investigation into the structural features and catalytic characterization of cold-adapted AhpC. The structural characteristics of PsAhpC and its cold tolerance and salt tolerance may be applied to stress resistance breeding of various organisms., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Extracellular proteases from haloarchaea, also referred to as halolysins, are in increasing demand and are studied for their various applications in condiments and leather industries. In this study, an extracellular protease encoding gene from the haloarchaeon Halorubellus sp. PRR65, hly65, was cloned and heterologously expressed in E. coli. The novel halolysin Hly65 from the genus Halorubellus was characterized by complete inhibition of phenylmethanesulfonyl fluoride (PMSF) on its enzyme activity. Experimental determination revealed a triad catalytic active center consisting of Asp 154 -His 193 -Ser 348 . Deletion of the C-terminal extension (CTE) resulted in loss of enzyme activity, while dithiothreitol (DTT) did not inhibit the enzyme activity, suggesting that Hly65 may function as a monomer. The K m , V max and K cat for the Hly65 were determined to be 2.91 mM, 1230.47 U·mg -1 and 1538.09 S -1 , respectively, under 60 °C, pH 8.0 and 4.0 M NaCl using azocasecin as a substrate. Furthermore, a three-dimensional structure prediction based on functional domains was obtained in this study which will facilitate modification and reorganization of halolysins to generate mutants with new physiological activities., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Leishmania braziliensis is a pathogenic protozoan parasite that causes American Tegumentary Leishmaniasis (ATL), an important tropical neglected disease. ENTPDases are nucleotidases that hydrolyze intracellular and/or extracellular nucleotides. ENTPDases are known as regulators of purinergic signalling induced by extracellular nucleotides. Leishmania species have two isoforms of ENTPDase, and, particularly, ENTPDase2 seems to be involved in infectivity and virulence. In this study, we conducted the heterologous expression and biochemical characterization of the recombinant ENTPDase2 of L. braziliensis (rLbNTPDase2). Our results show that this enzyme is a canonical ENTPDase with apyrase activity, capable of hydrolysing triphosphate and diphosphate nucleotides, and it is dependent on divalent cations (calcium or magnesium). Substrate specificity was characterized as UDP>GDP>ADP>GTP>ATP=UTP. The enzyme showed optimal activity at a neutral to basic pH and was partially inhibited by suramin and DIDS. Furthermore, the low apparent Km for ADP suggests that the enzyme may play a role in adenosine-mediated signalling. The biochemical characterization of this enzyme can open new avenues for using LbNTPDase2 as a drug target., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Glycans containing fucose play crucial roles in cell biology, particularly in recognition processes. In humans, fucose found in H-blood group antigens is recognized by various pathogens, thereby influencing host-pathogen interactions. However, in invertebrate biology the specific functions of these modifications and the corresponding glycosyltransferases are not fully elucidated. Therefore, cloning these glycosyltransferases from different model systems will provide valuable insights into this process. Little is known about fucosyltransferases in molluscs. For this study, a sequence of the Pacific oyster, Crassostrea gigas, based on amino acid sequence homologies with rabbit and human α-1,2-fucosyltransferases, was chosen. The recombinant enzyme (350 amino acids) was able to transfer fucose from GDP-fucose to the galactose residue of type II disaccharides, terminal galactoses in complex N-glycan structures and several linear and branched galactans which were tested using a glycan microarray. The α-1,2-linkage formed was confirmed by NMR analysis. The enzyme was active in a broad pH-range, it was relatively stable upon storage conditions and its activity was not dependent on the presence of divalent cations. In this study, we were able to clone, express and characterise a novel α-1,2-fucosyltrasferase from Crassostrea gigas (CgFUT2)., (© 2024. The Author(s).)

Key Message: Enhanced recombinant protein expression was achieved in Salinas lettuce and commercial lettuce by designing a unique RNAi that knockdown the gene-silencing mechanism in transient assays. Improved yields of recombinant proteins (RP) are necessary for protein-production efficiency and ease of purification. Achieving high yield in non-tobacco plants will enable diverse plants to be used as hosts in transient protein-expression systems. With improved protein yield, lettuce (Lactuca sativa) could take the lead as a plant host for RP production. Therefore, this study aimed to improve RP production in lettuce var. Salinas by designing a single RNA interference (RNAi) construct targeting LsRDR1 and LsRDR6 using the Tsukuba system vector. Two RNAi constructs, RNAi-1 and RNAi-2, targeting common regions of LsRDR1 and LsRDR6 with 75% and 76% similarity, respectively, were employed to evaluate simultaneous gene silencing. Quantitative transcription analysis demonstrated that both RNAi constructs effectively knocked down LsRDR6 and LsRDR1, but not LsRDR2, at both 3 and 5 days post-infiltration (dpi), with RNAi-1 exhibited slightly higher efficiency. Based on the protein yield, co-expression of RNAi-1 with enhanced green fluorescent protein (EGFP) increased EGFP expression by approximately 4.9-fold and 3.7-fold at 3 dpi and 5 dpi, respectively, compared to control. A similar but slightly lower increase (2.4-fold and 2.33-fold) was observed in commercial lettuce at 3 and 5 dpi, respectively. To confirm these results, co-infiltration with Bet v 1, a major allergen from birch pollen, resulted in a 2.5-fold increase in expression in Salinas lettuce at 5 dpi. This study marks a significant advancement in enhancing transient protein production in lettuce, elevating its potential as a host for recombinant protein production., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

High fidelity DNA polymerase from Pyrococcus furiosus (Pfupol) is an attractive alternative to the highly popular DNA polymerase from Thermus aquaticus. Because this enzyme is in great demand for biotechnological applications, optimizing Pfupol production is essential to supplying the industry's expanding demand. T7-induced promoter expression in Escherichia coli expression systems is used to express recombinant Pfupol; however, this method is not cost-effective. Here, we have effectively developed an optimized process for the autoinduction approach of Pfupol expression in a defined medium. To better examine Pfupol's activities, its purified fraction was used. A 71 mg/L of pure Pfupol was effectively produced, resulting in a 2.6-fold increase in protein yield when glucose, glycerol, and lactose were added in a defined medium at concentrations of 0.05%, 1%, and 0.6%, respectively, and the condition for production in a 5 L bioreactor was as follow: 200 rpm, 3 vvm, and 10% inoculant. Furthermore, the protein exhibited 1445 U/mg of specific activity when synthesized in its active state. This work presents a high level of Pfupol production, which makes it an economically viable and practically useful approach., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The natural source of chymosin, a key enzyme in the dairy industry, is insufficient for rapidly growing cheese industries. Large-scale production of recombinant proteins in heterologous hosts provides an efficient alternative solution. Here, the codon-optimized synthetic prochymosin gene, which has a CAI index of 0.926, was subcloned from a cloning vector (pUC57-bCYM) into the pBI121 vector, resulting in the construct named pBI121-bCYM. CAI ranges from 0 to 1 and higher CAI improves gene expression in heterologous hosts. The overexpression of the prochymosin gene was under the control of constitutive CaMV 35S promoter and NOS terminator and was transferred into the tobacco via A. tumefaciens strain LBA4404. Explant type, regeneration method, inoculation temperature, cell density (OD 600 ) of Agrobacterium for inoculation, and acetosyringone concentration were leaf explants, direct somatic embryogenesis, 19 °C, 0.1, and 100 µM, respectively. The successful integration and expression of the prochymosin gene, along with the bioactivity of recombinant chymosin, were confirmed by PCR, RT-PCR, and milk coagulation assay, respectively. Overall, this study reports the first successful overexpression of the codon-optimized prochymosin form of the bovine chymosin enzyme in the tobacco via indirect transformation. Production of recombinant bovine chymosin in plants can be an easy-to-scale-up, safe, and inexpensive platform., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Chitinases are enzymes that can break down chitin, a major component of the exoskeleton of insects and fungi. This feature makes them potential biopesticides in agriculture since they are considered a safe and environmentally friendly alternative to synthetic pesticides. In this work, we performed a comparative study between two different bacterial expression strains to produce a recombinant chitinase with improved stability. Escherichia coli strains Origami B and BL21 (DE3) were selected for their distinct cytosolic environment to express BhChitA chitinase of Bacillus halodurans C-125 and to investigate the role of disulfide bond formation and proper folding on its stability and activity. Expression of the recombinant BhChitA in bacterial strain containing oxidative cytosol (Origami B) improved its activity and stability. Although both expression systems have comparable biochemical properties (temperature range 20-80 °C and pH spectrum 3-10), BhChitA expressed in Origami strain seems more stable than expressed in BL21. Furthermore, the optimal expression conditions of the recombinant BhChitA has been carried out at 30 °C during 6 h for the Origami strain, against 20 °C during 2 h for BL21. On the other hand, no significant differences were detected between the two enzymes when the effect of metal ions was tested. These findings correlate with the analysis of the overall structure of BhChitA. The model structure permitted to localize disulfide bond, which form a stable connection between the substrate-binding residues and the hydrophobic core. This link is required for efficient binding of the chitin insertion domain to the substrate. BhChitA exhibited in vitro antifungal effect against phytopathogenic fungi and suppressed necrosis of Botrytis cinerea on detached tomato leaves. In vitro assays showed the influence of BhChitA on growth suppression of Botrytis cinerea (53%) Aspergillus niger (65%), Fusarium graminearum (25%), and Fusarium oxysporum (34%). Our results highlight the importance of the bacterial expression system with oxidative cytosol in producing promising biopesticides that can be applied for post-harvest processing and crop protection., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

L-asparaginase is an essential enzyme used in cancer treatment, but its production faces challenges like low yield, high cost, and immunogenicity. Recombinant production is a promising method to overcome these limitations. In this study, response surface methodology (RSM) was used to optimize the production of L-asparaginase 1 from Saccharomyces cerevisiae in Escherichia coli K-12 BW25113. The Box-Behnken design (BBD) was utilized for the RSM modeling, and a total of 29 experiments were conducted. These experiments aimed to examine the impact of different factors, including the concentration of isopropyl-b-LD-thiogalactopyranoside (IPTG), the cell density prior to induction, the duration of induction, and the temperature, on the expression level of L-asparaginase 1. The results revealed that while the post-induction temperature, cell density at induction time, and post-induction time all had a significant influence on the response, the post-induction time exhibited the greatest effect. The optimized conditions (induction at cell density 0.8 with 0.7 mM IPTG for 4 h at 30 °C) resulted in a significant amount of L-asparaginase with a titer of 93.52 μg/mL, which was consistent with the model-based prediction. The study concluded that RSM optimization effectively increased the production of L-asparaginase 1 in E. coli, which could have the potential for large-scale fermentation. Further research can explore using other host cells, optimizing the fermentation process, and examining the effect of other variables to increase production., (© 2024. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

A novel β-galactosidase gene (PbBgal35A) from Pedobacter sp. CAUYN2 was cloned and expressed in Escherichia coli. The gene had an open reading frame of 1917 bp, encoding 638 amino acids with a predicted molecular mass of 62.3 kDa. The deduced amino acid sequence of the gene shared the highest identity of 41% with a glycoside hydrolase family 35 β-galactosidase from Xanthomonas campestris pv. campestris (AAP86763.1). The recombinant β-galactosidase (PbBgal35A) was purified to homogeneity with a specific activity of 65.9 U/mg. PbBgal35A was optimally active at pH 5.0 and 50 °C, respectively, and it was stable within pH 4.5‒7.0 and up to 45 °C. PbBgal35A efficiently synthesized galacto-oligosaccharides from lactose with a conversion ratio of 32% (w/w) and fructosyl-galacto-oligosaccharides from lactulose with a conversion ratio of 21.9% (w/w). Moreover, the enzyme catalyzed the synthesis of galacto-oligosaccharides from low-content lactose in fresh milk, and the GOS conversion ratios of 17.1% (w/w) and 7.8% (w/w) were obtained when the reactions were performed at 45 and 4 °C, respectively. These properties make PbBgal35A an ideal candidate for commercial use in the manufacturing of GOS-enriched dairy products., (© 2024. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

Lipases are industrially important enzymes having vast applications in various fields. Cloning and expression of lipase enzyme-encoding genes in suitable host lead to their widespread use in different fields. The present study represents the first attempt towards the expression of the synthetic lipase gene in Pseudomonas aeruginosa. An alkalophilic lipase gene (GenBank accession number: NP&#95;388152) from Bacillus subtilis was synthetically designed and introduced in the pJN105 vector and subsequently cloned in Pseudomonas aeruginosa SDK-6. Agarose gel electrophoresis confirmed the transformation of SDK-6, exhibiting a band difference of ~ 700 bp between native and recombinant pJN105. Further amplification of cloned lipase gene was confirmed using PCR amplification with Lip 1 and Lip 2 primers respectively, followed by restriction analysis. Approximately 15-fold increase in lipase production was observed in recombinant Pseudomonas as compared to the native strain. One factor at a time (OFAT) analysis revealed L-arabinose, inoculum size (0.5%; v/v), and agitation (120 rpm) as significant factors affecting the over-expression of lipase enzyme. Optimization of enzyme induction conditions by central composite design (CCD) led to 1.60-fold increase in the production of lipase at 0.65% (w/v) inducer concentration, OD 600 -1.075 before induction and 35 °C post induction temperature with overall lipase production of 50.50 IU/mL. Statistical validation of observed value via ANOVA showed an F-value of 138.70 at p < 0.01 with R 2 of 0.9921., (© 2024. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

Dye-decolorizing peroxidases (DyPs) belong to a novel superfamily of heme peroxidases that can oxidize recalcitrant compounds. In the current study, the GlDyP2 gene from Ganoderma lucidum was heterologously expressed in Escherichia coli, and the enzymatic properties of the recombinant GlDyP2 protein were investigated. The GlDyP2 protein could oxidize not only the typical peroxidase substrate ABTS but also two lignin substrates, namely guaiacol and 2,6-dimethoxy phenol (DMP). For the ABTS substrate, the optimum pH and temperature of GlDyP2 were 4.0 and 35 °C, respectively. The pH stability and thermal stability of GlDyP2 were also measured; the results showed that GlDyP2 could function normally in the acidic environment, with a T 50 value of 51 °C. Moreover, compared to untreated controls, the activity of GlDyP2 was inhibited by 1.60 mM of Mg 2+ , Ni 2+ , Mn 2+ , and ethanol; 0.16 mM of Cu 2+ , Zn 2+ , methanol, isopropyl alcohol, and Na 2 EDTA·2H 2 O; and 0.016 mM of Fe 2+ and SDS. The kinetic constants of recombinant GlDyP2 for oxidizing ABTS, Reactive Blue 19, guaiacol, and DMP were determined; the results showed that the recombination GlDyP2 exhibited the strongest affinity and the most remarkable catalytic efficiency towards guaiacol in the selected substrates. GlDyP2 also exhibited decolorization and detoxification capabilities towards several dyes, including Reactive Blue 19, Reactive Brilliant Blue X-BR, Reactive Black 5, Methyl Orange, Trypan Blue, and Malachite Green. In conclusion, GlDyP2 has good application potential for treating dye wastewater., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg 2+ binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg 2+ . Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg 2+ , while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg 2+ increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg 2+ . Conclusively, the presence of Mg 2+ induces a conformation in KpPrAT that favours nucleotide binding., (© 2024. The Author(s).)

To investigate the effect of α3 and α5 helices on the biochemical characterization of Bacillus thermocatenulatus lipase (BTL2), both helices were deleted from native BTL2 lipase. After structural modeling and characterization, the truncated btl2 gene (Δbtl2) was cloned into E. coli BL21 under the control of the T7 promoter. After cultivation and induction of the recombinant bacteria, the Δα3α5 lipase was purified by Ni-NTA column chromatography. Next, the biochemical properties of the Δα3α5 lipase were compared with the previously expressed and purified native lipase. In the presence of the substrate tributyrin (C4), the maximum activity of native and Δα3α5 lipase was 9360 and 5000 U/mg, respectively. The deletion changed the substrate specificity from tributyrin (C4) to tricaprylin (C8) substrate. Native and Δα3α5 lipase showed similar activity patterns at all temperatures and pH values, with the activity of Δα3α5 lipase being approximately 20% lower than native lipase. Triton X100 increased the activity of native and Δα3α5 lipases by 2.1- and 2.5-fold, respectively., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

This study aims to highlight the potential use of cTNAs in therapeutic applications. The COVID-19 pandemic has led to significant use of coding therapeutic nucleic acids (cTNAs) in terms of DNA and mRNA in the development of vaccines. The use of cTNAs resulted in a paradigm shift in the therapeutic field. However, the injection of DNA or mRNA into the human body transforms cells into biological factories to produce the necessary proteins. Despite the success of cTNAs in the production of corona vaccines, they have several limitations such as instability, inability to cross biomembranes, immunogenicity, and the possibility of integration into the human genome. The chemical modification and utilization of smart drug delivery cargoes resolve cTNAs therapeutic problems. The success of cTNAs in corona vaccine production provides perspective for the eradication of influenza viruses, Zika virus, HIV, respiratory syncytial virus, Ebola virus, malaria, and future pandemics by quick vaccine design. Moreover, the progress cTNAs technology is promising for the development of therapy for genetic disease, cancer therapy, and currently incurable diseases., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Key Message: E1 holoenzyme was extensively Hyp-O-glycosylated at the proline rich linker region in plants, which substantially increased the molecular size and improved the enzymatic digestibility of the biomass of transgenic plants. Thermophilic E1 endo-1,4-β-glucanase derived from Acidothermus cellulolyticus has been frequently expressed in planta to reconstruct the plant cell wall to overcome biomass recalcitrance. However, the expressed holoenzyme exhibited a larger molecular size (~ 100 kDa) than the theoretical one (57 kDa), possibly due to posttranslational modifications in the recombinant enzyme within plant cells. This study investigates the glycosylation of the E1 holoenzyme expressed in tobacco plants and determines its impact on enzyme activity and biomass digestibility. The E1 holoenzyme, E1 catalytic domain (E1cd) and E1 linker (E1Lk) were each expressed in tobacco plants and suspension cells. The accumulation of holoenzyme was 2.0- to 2.3- times higher than that of E1cd. The proline-rich E1Lk region was extensively hydroxyproline-O-glycosylated with arabinogalactan polysaccharides. Compared with E1cd, the holoenzyme displayed a broader optimal temperature range (70 to 85 ºC). When grown in greenhouse, the expression of E1 holoenzyme induced notable phenotypic changes in plants, including delayed flowering and leaf variegation post-flowering. However, the final yield of plant biomass was not significantly affected. Finally, plant biomass engineering with E1 holoenzyme showed 1.7- to 1.8-fold higher saccharification efficiency than the E1cd lines and 2.4- to 2.7-fold higher than the wild-type lines, which was ascribed to the synergetic action of the E1Lk and cellulose binding module in reducing cell wall recalcitrance., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The methylotrophic yeast Pichia pastoris is garnering interest as a chassis cell factory for the manufacture of recombinant proteins because it effectively satisfies the requirements of both laboratory and industrial set up. The optimisation of P. pastoris cultivation is still necessary due to strain- and product-specific problems such as promoter strength, methanol utilisation type, and culturing conditions to realize the high yields of heterologous protein(s) of interest. Techniques integrating genetic and process engineering have been used to overcome these problems. Insight into the Pichia as an expression system utilizing MUT pathway and the development of methanol free systems are highlighted in this systematic review. Recent developments in the improved production of proteins in P. pastoris by (i) diverse genetic engineering such as codon optimization and gene dosage; (ii) cultivating tactics including co-expression of chaperones; (iii) advances in the use of the 2A peptide system, and (iv) CRISPR/Cas technologies are widely discussed. We believe that by combining these strategies, P. pastoris will become a formidable platform for the production of high value therapeutic proteins., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Aeromonas hydrophila, an opportunistic warm water pathogen, has always been a threat to aquaculture, leading to substantial economic losses. Vaccination of the cultured fish would effectively prevent Aeromoniasis, and recent advancements in nanotechnology show promise for efficacious vaccines. Oral delivery would be the most practical and convenient method of vaccine delivery in a grow-out pond. This study studied the immunogenicity and protective efficacy of a nanoparticle-loaded outer membrane protein A from A. hydrophila in the zebrafish model. The protein was over-expressed, purified, and encapsulated using poly lactic-co-glycolic acid (PLGA) nanoparticles via the double emulsion method. The PLGA nanoparticles loaded with recombinant OmpA (rOmpA) exhibited a size of 295 ± 15.1 nm, an encapsulation efficiency of 72.52%, and a polydispersity index of 0.292 ± 0.07. Scanning electron microscopy confirmed the spherical and isolated nature of the PLGA-rOmpA nanoparticles. The protective efficacy in A. hydrophila-infected zebrafish after oral administration of the nanovaccine resulted in relative percentage survival of 77.7. Gene expression studies showed significant upregulation of immune genes in the vaccinated fish. The results demonstrate the usefulness of oral administration of nanovaccine-loaded rOmpA as a potential vaccine since it induced a robust immune response and conferred adequate protection against A. hydrophila in zebrafish, Danio rerio., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Disintegrins, a family of snake venom protein, which are capable of modulating the activity of integrins that play a fundamental role in the regulation of many physiological and pathological processes. The main purpose of this study is to obtain the recombinant disintegrin (r-DI) and evaluate its biological activity. In this study, we explored a high-level expression prokaryotic system and purification strategy for r-DI. Then, r-DI was treated to assay effects on cell growth, migration, and invasion. The affinity for the interactions of r-DI with integrin was determined using Surface plasmon resonance (SPR) analyses. The r-DI can be expressed in Escherichia coli and purified by one-step chromatography. The r-DI can inhibit B16F10 cells proliferation, migration, and invasion. Also, we found that r-DI could interact with the integrin αIIbβ3 (GPIIb/IIIa). The r-DI can be expressed, purified, characterized through functional assays, and can also maintain strong biological activities. Thus, this study showed potential therapeutic effects of r-DI for further functional and structural studies., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

We compared the immunogenicity of recombinant S. pneumoniae pneumolysin (rPly) when administered with and without Al(OH) 3 adjuvant, and evaluated the protective properties of recombinant protein in the active defense experiment. It was shown that double immunization with rPly+Al(OH) 3 increases the levels of IgG antibodies in comparison with the control (p<0.01), while triple immunization results in a more significant increase in IgG antibody levels (p<0.001). Double immunization with rPly without Al(OH) 3 does not induce a significant increase in antibody levels in comparison with the control, while triple immunization results in a slight but significant increase in antibody levels (p<0.05). The active defense test proved the protective activity of rPly against S. pneumoniae serotype 3 at intranasal infection., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Stonustoxin (SNTX) is a lethal protein found in stonefish venom, responsible for many of the symptoms associated with stonefish envenomation. To counter stonefish venom challenges, antivenom is a well-established and effective solution. In this study, we aimed to produce the recombinant alpha subunit protein of Stonustoxin from Synanceia horrida and prepare antibodies against it The SNTXα gene sequence was optimized for E. coli BL21 (DE3) expression and cloned into the pET17b vector. Following purification, the recombinant protein was subcutaneously injected into rabbits, and antibodies were extracted from rabbit´s serum using a G protein column As a result of codon optimization, the codon adaptation index for the SNTXα cassette increased to 0.94. SDS-PAGE analysis validated the expression of SNTXα, with a band observed at 73.5 kDa with a yield of 60 mg/l. ELISA results demonstrated rabbits antibody titers were detectable up to a 1:256,000 dilution. The isolated antibody from rabbit´s serum exhibited a concentration of 1.5 mg/ml, and its sensitivity allowed the detection of a minimum protein concentration of 9.7 ng. In the neutralization assay the purified antibody against SNTXα protected mice challenged with 2 LD50. In conclusion, our study successfully expressed the alpha subunit of Stonustoxin in a prokaryotic host, enabling the production of antibodies for potential use in developing stonefish antivenom., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Antimicrobial peptides or bacteriocins are excellent candidates for alternative antimicrobials, but high manufacturing costs limit their applications. Recombinant gene expression offers the potential to produce these peptides more cost-effectively at a larger scale. Saccharomyces cerevisiae is a popular host for recombinant protein production, but with limited success reported on antimicrobial peptides. Individual recombinant S. cerevisiae strains were constructed to secrete two class IIa bacteriocins, plantaricin 423 (PlaX) and mundticin ST4SA (MunX). The native and codon-optimised variants of the plaA and munST4SA genes were cloned into episomal expression vectors containing either the S. cerevisiae alpha mating factor (MFα1) or the Trichoderma reesei xylanase 2 (XYNSEC) secretion signal sequences. The recombinant peptides retained their activity and stability, with the MFα1 secretion signal superior to the XYNSEC secretion signal for both bacteriocins. An eight-fold increase in activity against Listeria monocytogenes was observed for MunX after codon optimisation, but not for PlaX-producing strains. After HPLC-purification, the codon-optimised genes yielded 20.9 mg/L of MunX and 18.4 mg/L of PlaX, which displayed minimum inhibitory concentrations (MICs) of 108.52 nM and 1.18 µM, respectively, against L. monocytogenes. The yields represent a marked improvement relative to an Escherichia coli expression system previously reported for PlaX and MunX. The results demonstrated that S. cerevisiae is a promising host for recombinant bacteriocin production that requires a simple purification process, but the efficacy is sensitive to codon usage and secretion signals., (© 2023. The Author(s).)

Glutathione-S-transferase enzymes (GSTs) are essential components of the phase II detoxification system and protect organisms from oxidative stress induced by xenobiotics and harmful toxins such as 1-chloro-2,4-dinitrobenzene (CDNB). In Tetrahymena thermophila, the TtGSTm34 gene was previously reported to be one of the most responsive GST genes to CDNB treatment (LD50 = 0.079 mM). This study aimed to determine the kinetic features of recombinantly expressed and purified TtGSTm34 with CDNB and glutathione (GSH). TtGSTm34-8xHis was recombinantly produced in T. thermophila as a 25-kDa protein after the cloning of the 660-bp full-length ORF of TtGSTm34 into the pIGF-1 vector. A three-dimensional model of the TtGSTm34 protein constructed by the AlphaFold and PyMOL programs confirmed that it has structurally conserved and folded GST domains. The recombinant production of TtGSTm34-8xHis was confirmed by SDS‒PAGE and Western blot analysis. A dual-affinity chromatography strategy helped to purify TtGSTm34-8xHis approximately 3166-fold. The purified recombinant TtGSTm34-8xHis exhibited significantly high enzyme activity with CDNB (190 µmol/min/mg) as substrate. Enzyme kinetic analysis revealed K m values of 0.68 mM with GSH and 0.40 mM with CDNB as substrates, confirming its expected high affinity for CDNB. The optimum pH and temperature were determined to be 7.0 and 25 °C, respectively. Ethacrynic acid inhibited fully TtGSTm34-8xHis enzyme activity. These results imply that TtGSTm34 of T. thermophila plays a major role in the detoxification of xenobiotics, such as CDNB, as a first line of defense in aquatic protists against oxidative damage., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The β-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the β-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the β-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity., (© 2024. The Author(s).)

Cariogenic biofilms have a matrix rich in exopolysaccharides (EPS), mutans and dextrans, that contribute to caries development. Although several physical and chemical treatments can be employed to remove oral biofilms, those are only partly efficient and use of biofilm-degrading enzymes represents an exciting opportunity to improve the performance of oral hygiene products. In the present study, a member of a glycosyl hydrolase family 66 from Flavobacterium johnsoniae (FjGH66) was heterologously expressed and biochemically characterized. The recombinant FjGH66 showed a hydrolytic activity against an early EPS-containing S. mutans biofilm, and, when associated with a α-(1,3)-glucosyl hydrolase (mutanase) from GH87 family, displayed outstanding performance, removing more than 80% of the plate-adhered biofilm. The mixture containing FjGH66 and Prevotella melaninogenica GH87 α-1,3-mutanase was added to a commercial mouthwash liquid to synergistically remove the biofilm. Dental floss and polyethylene disks coated with biofilm-degrading enzymes also degraded plate-adhered biofilm with a high efficiency. The results presented in this study might be valuable for future development of novel oral hygiene products., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Recombinant protein production technology is widely applied to the manufacture of biologics used as drug substances and industrial proteins such as recombinant enzymes and bioactive proteins. Various heterologous protein production systems have been developed using prokaryotic and eukaryotic hosts. Especially methylotrophic yeast in eukaryotic hosts is suggested to be particularly valuable because such systems have the following advantages: protein secretion into culture broth, eukaryotic quality control systems, a post-translational modification system, rapid growth, and established recombinant DNA tools and technologies such as strong promoters, effective selection markers, and gene knock-in and -out systems. Many methylotrophic yeasts such as the genera Candida, Ogataea, and Komagataella have been studied since methylotrophic yeast was first isolated in 1969. The methanol-consumption-related genes in methylotrophic yeast are strongly and strictly regulated under methanol-containing conditions. The well-regulated gene expression systems under the methanol-inducible gene promoter lead to the potential application of heterologous protein production in methylotrophic yeast. In this review, we describe the recent progress of heterologous protein production technology in methylotrophic yeast and introduce Ogataea minuta as an alternative production host as a substitute for K. phaffii and O. polymorpha., (© 2024. The Author(s).)

Glucagon-like peptide-1(GLP-1) is an incretin hormone secreted primarily from the intestinal L-cells in response to meals. GLP-1 is a key regulator of energy metabolism and food intake. It has been proven that P9 protein from A. muciniphila could increase GLP-1 release and improve glucose homeostasis in HFD-induced mice. To obtain an engineered Lactococcus lactis which produced P9 protein, mature polypeptide chain of P9 was codon-optimized, fused with N-terminal signal peptide Usp45, and expressed in L. lactis NZ9000. Heterologous secretion of P9 by recombinant L. lactis NZP9 were successfully detected by SDS-PAGE and western blotting. Notably, the supernatant of L. lactis NZP9 stimulated GLP-1 production of NCI-H716 cells. The relative expression level of GLP-1 biosynthesis gene GCG and PCSK1 were upregulated by 1.63 and 1.53 folds, respectively. To our knowledge, this is the first report on the secretory expression of carboxyl-terminal processing protease P9 from A. muciniphila in L. lactis. Our results suggest that genetically engineered L. lactis which expressed P9 may have therapeutic potential for the treatment of diabetes, obesity and other metabolic disorders., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Bacillus subtilis is regarded as a promising microbial expression system in bioengineering due to its high stress resistance, nontoxic, low codon preference and grow fast. The strain has a relatively efficient expression system, as it has at least three protein secretion pathways and abundant molecular chaperones, which guarantee its expression ability and compatibility. Currently, many proteins are expressed in Bacillus subtilis, and their application prospects are broad. Although Bacillus subtilis has great advantages compared with other prokaryotes related to protein expression and secretion, it still faces deficiencies, such as low wild-type expression, low product activity, and easy gene loss, which limit its large-scale application. Over the years, many researchers have achieved abundant results in the modification of Bacillus subtilis expression systems, especially the optimization of promoters, expression vectors, signal peptides, transport pathways and molecular chaperones. An optimal vector with a suitable promoter strength and other regulatory elements could increase protein synthesis and secretion, increasing industrial profits. This review highlights the research status of optimization strategies related to the expression system of Bacillus subtilis. Moreover, research progress on its application as a food-grade expression system is also presented, along with some future modification and application directions., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

We studied toxicity of recombinant Streptococcus pneumoniae pneumolysin protein in experiments on mice and its cytopathogenic effect on cultures of Vero green monkey kidney cells and human lung carcinoma A549 cells in vitro. In vivo and in vitro experiments proved the absence of compromised toxicity and direct cytopathogenic action of the recombinant protein., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Most plant and bacterial toxins are highly immunogenic with non-specific toxic effects. Human ribonucleases are thought to provide a promising basis for reducing the toxic agent's immunogenic properties, which are candidates for cancer therapy. In the cell, the ribonuclease inhibitor (RI) protein binds to the ribonuclease enzyme and forms a tight complex. This study aimed to engineer and provide a gene construct encoding an improved version of Human Pancreatic RNase 1 (HP-RNase 1) to reduce connection to RI and modulate the immunogenic effects of immunotoxins. To further characterize the interaction complex of HP-RNase 1 and RI, we established various in silico and in vitro approaches. These methods allowed us to specifically monitor interactions within native and engineered HP-RNase 1/RI complexes. In silico research involved molecular dynamics (MD) simulations of native and mutant HP-RNase 1 in their free form and when bound to RI. For HP-RNase 1 engineering, we designed five mutations (K8A/N72A/N89A/R92D/E112/A) based on literature studies, as this combination proved effective for the intended investigation. Then, the cDNA encoding HP-RNase 1 was generated by RT-PCR from blood and cloned into the pSYN2 expression vector. Consequently, wild-type and the engineered HP-RNase 1 were over-expressed in E. coli TG1 and purified using an IMAC column directed against a poly-his tag. The protein products were detected by SDS-PAGE and Western blot analysis. HP-RNase 1 catalytic activity, in the presence of various concentrations of RI, demonstrated that the mutated version of the protein is able to escape the ribonuclease inhibitor and target the RNA substrate 2.5 folds more than that of the wild type. From these data, we tend to suggest the engineered recombinant HP-RNase 1 potentially as a new immunotherapeutic agent for application in human cancer therapy., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Scavenger receptors are a protein superfamily that typically consists of one or more repeats of the scavenger receptor cysteine-rich structural domain (SRCRD), which is an ancient and highly conserved protein module. The expression and purification of eukaryotic proteins containing multiple disulfide bonds has always been challenging. The expression systems that are commonly used to express SRCRD proteins mainly consist of eukaryotic protein expression systems. Herein, we established a high-level expression strategy of a Type B SRCRD unit from human salivary agglutinin using the Escherichia coli expression system, followed by a refolding and purification process. The untagged recombinant SRCRD was expressed in E. coli using the pET-32a vector, which was followed by a refolding process using the GSH/GSSG redox system. The SRCRD expressed in E. coli SHuffle T7 showed better solubility after refolding than that expressed in E. coli BL21(DE3), suggesting the importance of the disulfide bond content prior to refolding. The quality of the refolded protein was finally assessed using crystallization and crystal structure analysis. As proteins refolded from inclusion bodies exhibit a high crystal quality and reproducibility, this method is considered a reliable strategy for SRCRD protein expression and purification. To further confirm the structural integrity of the refolded SRCRD protein, the purified protein was subjected to crystallization using sitting-drop vapor diffusion method. The obtained crystals of SRCRD diffracted X-rays to a resolution of 1.47 Å. The solved crystal structure appeared to be highly conserved, with four disulfide bonds appropriately formed. The surface charge distribution of homologous SRCRD proteins indicates that the negatively charged region at the surface is associated with their calcium-dependent ligand recognition. These results suggest that a high-quality SRCRD protein expressed by E. coli SHuffle T7 can be successfully folded and purified, providing new options for the expression of members of the scavenger receptor superfamily., (© 2024. The Author(s).)

Canine circovirus (CanineCV) is an emerging pathogen in domestic dogs, detected in multiple countries in association with varying clinical and pathological presentations including diarrhoea, vasculitis, granulomatous inflammation, and respiratory signs. Understanding the pathology of CanineCV is confounded by the fact that it has been detected in asymptomatic dogs as well as in diseased dogs concurrently infected with known pathogens. Recombinantly expressed self-assembling Virus-like particles (VLPs) lack viral genomic material but imitate the capsid surface conformations of wild type virion, allowing arrays of biological applications including subunit vaccine development and immunodiagnostics. In this study, full length CanineCV capsid gene was expressed in Escherichia coli followed by two-step purification process to yield soluble capsid protein in high concentration. Transmission electron microscopy (TEM) confirmed the capsid antigen self-assembled into 17-20 nm VLPs in glutathione S-transferase (GST) buffer, later utilised to develop an indirect enzyme-linked immunosorbent assay (iELISA). The respective sensitivity and specificity of the proposed iELISA were 94.10% and 88.40% compared with those obtained from Western blot. The mean OD 450 value for western blot positive samples was 1.22 (range 0.12-3.39) and negative samples was 0.21 (range 0.07-0.41). An optimal OD 450 cut-off of 0.35 was determined by ROC curve analysis. Median inter-assay and intra-assay validation revealed that the iELISA test results were reproducible with coefficients of variation 7.70 (range 5.6-11.9) and 4.21 (range 1.2-7.4). Our results demonstrated that VLP-based iELISA is a highly sensitive method for serological diagnosis of CanineCV infections in dogs, suitable for large-scale epidemiological studies., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Heavy and irresponsible use of antibiotics in the last century has put selection pressure on the microbes to evolve even faster and develop more resilient strains. In the confrontation with such sometimes called "superbugs", the search for new sources of biochemical antibiotics seems to have reached the limit. In the last two decades, bioactive antimicrobial peptides (AMPs), which are polypeptide chains with less than 100 amino acids, have attracted the attention of many in the control of microbial pathogens, more than the other types of antibiotics. AMPs are groups of components involved in the immune response of many living organisms, and have come to light as new frontiers in fighting with microbes. AMPs are generally produced in minute amounts within organisms; therefore, to address the market, they have to be either produced on a large scale through recombinant DNA technology or to be synthesized via chemical methods. Here, heterologous expression of AMPs within bacterial, fungal, yeast, plants, and insect cells, and points that need to be considered towards their industrialization will be reviewed., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

This work aimed to assess the Sf9 cell metabolism during growth, and infection steps with recombinant baculovirus bearing rabies virus proteins, to finally obtain rabies VLP in two culture systems: Schott flask (SF) and stirred tank reactor (STR). Eight assays were performed in SF and STR (four assays in each system) using serum-free SF900 III culture medium. Two non-infection growth kinetics assays and six recombinant baculovirus infection assays. The infection runs were carried out at 0.1 pfu/cell multiplicity of infection (MOI) for single baculovirus bearing rabies glycoprotein (BVG) and matrix protein (BVM) and a coinfection with both baculoviruses at MOI of 3 and 2 pfu/cell for BVG and BVM, respectively. The SF assays were done in triplicate. The glucose, glutamine, glutamate, lactate, and ammonium uptake or release specific rates were quantified over the exponential growth phase and infection stage. The highest uptake specific rate was observed for glucose (42.5 × 10 -12  mmol cell/h) in SF and for glutamine (30.8 × 10 -12  mmol/cell/h) in STR, in the exponential growth phases. A wave pattern was observed for assessed analytes throughout the infection phase and the glucose had the highest wave amplitude within the 10 -10  mmol cell/h order. This alternative uptake and release behavior is in harmony with the lytic cycle of baculovirus in insect cells. The virus propagation and VLP generation were not limited by glucose, glutamine, and glutamate, neither by the toxicity of lactate nor ammonium under the conditions appraised in this work. The findings from this work can be useful to set baculovirus infection processes at high cell density to improve rabies VLP yield, purity, and productivity., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Lymphocyte function-associated antigene-1 (LFA-1) is a well-described integrin found on lymphocytes and other leukocytes, which is known to be overexpressed in leukemias and lymphomas. This receptor plays a significant role in immune responses such as T-cell activation, leukocyte cell-cell interactions, and trafficking of leukocyte populations. Subsequently, binders of LFA-1 emerge as potential candidates for cancer and autoimmune therapy. This study used the phage display technique to construct and characterize a high-affinity single-chain fragment variable (scFv) antibody against LFA-1. After expression, purification, dialysis, and concentration of the recombinant LFA-1 protein, four female BALB/c mice were immunized, splenocyte's mRNA was extracted, and cDNA was synthesized. A scFv library was constructed by linking the amplified V H /V κ fragments through a 72-bp linker using SOEing PCR. Next, the scFv gene fragments were cloned into the pComb-3XSS phagemid vector; thus, the phage library was developed. The selection process involved three rounds of phage-bio-panning, polyclonal, and monoclonal phage ELISA. AF17 was chosen and characterized among the positive clones through SDS-PAGE, Western blotting, indirect ELISA, and in-silico analyses. The results of the study showed the successful construction of a high-affinity scFv library against LFA-1. The accuracy of the AF17 production and its ability to bind to the LFA-1 were confirmed through SDS-PAGE, Western blot, and ELISA. This study highlights the potential application of the high-affinity AF17 against LFA-1 for targeting T lymphocytes for therapeutic purposes., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Inherited retinal dystrophies are often associated with mutations in the genes involved in the phototransduction cascade in photoreceptors, a paradigmatic signaling pathway mediated by G protein-coupled receptors. Photoreceptor viability is strictly dependent on the levels of the second messengers cGMP and Ca 2+ . Here we explored the possibility of modulating the phototransduction cascade in mouse rods using direct or liposome-mediated administration of a recombinant protein crucial for regulating the interplay of the second messengers in photoreceptor outer segments. The effects of administration of the free and liposome-encapsulated human guanylate cyclase-activating protein 1 (GCAP1) were compared in biological systems of increasing complexity (in cyto, ex vivo, and in vivo). The analysis of protein biodistribution and the direct measurement of functional alteration in rod photoresponses show that the exogenous GCAP1 protein is fully incorporated into the mouse retina and photoreceptor outer segments. Furthermore, only in the presence of a point mutation associated with cone-rod dystrophy in humans p.(E111V), protein delivery induces a disease-like electrophysiological phenotype, consistent with constitutive activation of the retinal guanylate cyclase. Our study demonstrates that both direct and liposome-mediated protein delivery are powerful complementary tools for targeting signaling cascades in neuronal cells, which could be particularly important for the treatment of autosomal dominant genetic diseases., (© 2023. The Author(s).)

Vibrio natriegens is a fast-growing, non-pathogenic marine bacterium with promising features for biotechnological applications such as high-level recombinant protein production or fast DNA propagation. A remarkable short generation time (< 10 min), robust proteosynthetic activity and versatile metabolism with abilities to utilise wide range of substrates contribute to its establishment as a future industrial platform for fermentation processes operating with high productivity.D,D-carboxypeptidases are membrane-associated enzymes involved in peptidoglycan biosynthesis and cell wall formation. This study investigates the impact of overexpressed D,D-carboxypeptidases on membrane integrity and the increased leakage of intracellular proteins into the growth medium in V. natriegens. Our findings confirm that co-expression of these enzymes can enhance membrane permeability, thereby facilitating the transport of target proteins into the extracellular environment, without the need for secretion signals, tags, or additional permeabilization methods. Using only a single step IMAC chromatography, we were able to purify AfKatG, MDBP or Taq polymerase in total yields of 117.9 ± 56.0 mg/L, 36.5 ± 12.9 mg/L and 26.5 ± 6.0 mg/L directly from growth medium, respectively. These results demonstrate the feasibility of our V. natriegens based system as a broadly applicable extracellular tag-less recombinant protein producer., (© 2023. The Author(s).)

The methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris) is a widely used host for extracellularly producing heterologous proteins via an expression cassette integrated into the yeast genome. A strong promoter in the expression cassette is not always the most favorable choice for heterologous protein production, especially if the correct folding of the protein and/or post-translational processing is the limiting step. The transcriptional terminator is another regulatory element in the expression cassette that can modify the expression levels of the heterologous gene. In this work, we identified and functionally characterized the promoter (P 1033 ) and transcriptional terminator (T 1033 ) of a constitutive gene (i.e., the 1033 gene) with a weak non-methanol-dependent transcriptional activity. We constructed two K. phaffii strains with two combinations of the regulatory DNA elements from the 1033 and AOX1 genes (i.e., P 1033 -T AOX1 and P 1033 -T 1033 pairs) and evaluated the impact of the regulatory element combinations on the transcript levels of the heterologous gene and endogenous 1033 and GAPDH genes in cells grown in glucose or glycerol, and on the extracellular product/biomass yield. The results indicate that the P 1033 has a 2-3% transcriptional activity of the GAP promoter and it is tunable by cell growth and the carbon source. The combinations of the regulatory elements rendered different transcriptional activity of the heterologous and endogenous genes that were dependent on the carbon source. The promoter-terminator pair and the carbon source affected the heterologous gene translation and/or protein secretion pathway. Moreover, low heterologous gene-transcript levels along with glycerol cultures increased translation and/or protein secretion., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Bacteriophage Phi11 harbors a gene, gp13, encoding the putative SSB protein (GenBank accession no. NC&#95;004615.1). SSB proteins bind to and protect the single-stranded DNA molecules from nuclease digestion and are essential for the growth and metabolic activities of the organisms encoding them. In this investigation, we have carried out the cloning, recombinant expression, and purification of rGp13 for the first time in Escherichia coli. EMSA data indicated that the purified recombinant Gp13 protein was capable of binding to single-stranded DNA. The protein exhibited maximum binding activity at 32 °C. Furthermore, our bioinformatic analysis has revealed that Gp13 consists of an OB-fold, a characteristic of SSB proteins. However, the arrangement of the OB-fold is unique, being located in the C-terminal domain of Gp13. Despite the importance of SSB proteins in various metabolic processes as well as in various types of PCR, there are no reports on the purification and characterization of SSB proteins from staphylococcal bacteriophages. We expect that the purification and characterization of recombinant Gp13 will help us gain a better insight into its biological activity and make it available in large quantities for molecular biology work., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

α-L-rhamnosidase [EC 3.2.1.40] belongs to glycoside hydrolase (GH) families (GH13, GH78, and GH106 families) in the carbohydrate-active enzymes (CAZy) database, which specifically hydrolyzes the non-reducing end of α-L-rhamnose. Αccording to the sites of catalytic hydrolysis, α-L-rhamnosidase can be divided into α-1, 2-rhamnosidase, α-1, 3-rhamnosidase, α-1, 4-rhamnosidase and α-1, 6-rhamnosidase. α-L-rhamnosidase is an important enzyme for various biotechnological applications, especially in food, beverage, and pharmaceutical industries. α-L-rhamnosidase has a wide range of sources and is commonly found in animals, plants, and microorganisms, and its microbial source includes a variety of bacteria, molds and yeasts (such as Lactobacillus sp., Aspergillus sp., Pichia angusta and Saccharomyces cerevisiae). In recent years, a series of advances have been achieved in various aspects of α-validates the above-described-rhamnosidase research. A number of α-L-rhamnosidases have been successfully recombinant expressed in prokaryotic systems as well as eukaryotic systems which involve Pichia pastoris, Saccharomyces cerevisiae and Aspergillus niger, and the catalytic properties of the recombinant enzymes have been improved by enzyme modification techniques. In this review, the sources and production methods, general and catalytic properties and biotechnological applications of α-L-rhamnosidase in different fields are summarized and discussed, concluding with the directions for further in-depth research on α-L-rhamnosidase., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

The IPTG-inducible promoter family, Pgrac, allows high protein expression levels in an inducible manner. In this study, we constructed IPTG-inducible expression vectors containing strong Pgrac promoters that allow integration of the transgene at either the amyE or lacA locus or both loci in Bacillus subtilis. Our novel integrative expression vectors based on Pgrac promoters could control the repression of protein production in the absence and the induction in the presence of an inducer, IPTG. The β-galactosidase (BgaB) protein levels were 9.0%, 15% and 30% of the total cellular protein in the B. subtilis strains carrying single cassettes with the Pgrac01, Pgrac100 or Pgrac212 promoters, respectively. The maximal induction ratio of Pgrac01-bgaB was 35.5 while that of Pgrac100-bgaB was 7.5 and that of Pgrac212-bgaB was 9. The inducible expression of GFP and BgaB protein was stably maintained for 24 h, with the highest yield of GFP being 24% of cell total protein while the maximum amount of BgaB was found to be 38%. A dual integration of two copies of the gfp + gene into the B. subtilis genome at the lacA and amyE loci resulted in a yield of about 40% of total cellular protein and a 1.74-fold increase in GFP compared with single-integrated strains containing the same Pgrac212 promoter. The capability of protein production from low to high levels of these inducible integrative systems is useful for fundamental and applied research in B. subtilis., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Autoinduction is a simple approach for heterologous protein expression that helps to achieve the high-level production of recombinant proteins in soluble form. In this work, we investigated if the application of an autoinduction strategy could help to optimize the production of bifunctional protein SRH-DR5-B, the DR5-specific TRAIL variant DR5-B fused to a VEGFR2-specific peptide SRHTKQRHTALH for dual antitumor and antiangiogenic activity. The protein was expressed in Escherichia coli SHuffle B T7, BL21(DE3), and BL21(DE3)pLysS strains. By IPTG induction, the highest expression level was in SHuffle B T7, while by autoinduction, the similar expression level was achieved in BL21(DE3)pLysS. However, in SHuffle B T7, only 45% of IPTG-induced SRH-DR5-B was expressed in soluble form, in contrast to 75% autoinduced in BL21(DE3)pLysS. The yield of purified SRH-DR5-B protein expressed by autoinduction in BL21(DE3)pLysS was 28 ± 4.5 mg per 200 ml of cell culture, which was 1.4 times higher than the yield from IPTG-induced SHuffle B T7. Regardless of the production method, SRH-DR5-B was equally cytotoxic to BxPC-3 human tumor cells expressing DR5 and VEGFR2 receptors. Thus, the production of SRH-DR5-B by autoinduction in the E. coli BL21(DE3)pLysS strain is an efficient, technologically simple, and economical technique that allows to obtain a large amount of active protein from the cytoplasmic cell fraction. Our work demonstrates that the strategy of induction of protein expression is no less important than the strain selection., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The production of vaccines in plant cells, termed plant-made pharmaceuticals or molecular farming, is a promising technology for scalable production. Compared to mammalian cell lines, like Chinese Hamster Ovary (CHO) or bacterial cells, plants can be grown with less cost on a large scale to make vaccines antigens and therapeutics affordable and accessible worldwide. An innovative application of this alternative system is the production of vaccines in edible tissues that can be consumed orally to deliver protein antigen without any further processing. In this project, we report stable expression of amino acid sequences corresponding to the TM-1 gene of Mycoplasma gallisepticum as a candidate vaccine antigen against Chronic Respiratory Disease (CRD) in chickens using wheat seed's tissues as a production host. Molecular and immunoblotting analysis confirmed the ubiquitous expression of a recombinant 41.8-kDa protein with an expression level of 1.03 mg/g dry weight in the endosperm tissues. When orally delivered, the plant-made vaccine was effective in terms of developing antibody response in animal model i.e., chicken without any detectable weight loss. Two doses of orally delivered plant-made TM-1 vaccine candidate elicited the immune response and protective effect against MG virus challenge at the level comparable to commercially available inactivated vaccine against CRD. Our study demonstrates that plant-made vaccines are not only safe but also scalable and cost-effective with prolonged stability at room temperature., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Pichia pastoris, a methylotrophic yeast, is known to be an efficient host for heterologous proteins production. In this study, a recombinant P. pastoris Y11430 was found better for β-glucosidase activity in comparison with a wild type P. pastoris Y11430 strain, and thereby, subjected to methanol intermittent feed profiling for β-glucosidase production. The results showed that at 72 h of cultivation time, the cultures with 16.67% and 33.33% methanol feeding with constant rate could produce the total dry cell weight of 52.23 and 118.55 g/L, respectively, while the total mutant β-glucosidase activities were 1001.59 and 3259.82 units, respectively. The methanol feeding profile was kept at 33% with three methanol feeding strategies such as constant feed rate, linear feed rate, and exponential feed rate which were used in fed-batch fermentation. At 60 h of cultivation, the highest total mutant β-glucosidase activity was 2971.85 units for exponential feed rate culture. On the other hand, total mutant β-glucosidase activity of the constant feed rate culture and linear feed rate culture were 1682.25 and 1975.43 units, respectively. The kinetic parameters of exponential feed rate culture were specific growth rate on glycerol 0.228/h, specific growth of methanol 0.061/h, maximum total dry cell weight 196.73 g, yield coefficient biomass per methanol ([Formula: see text]) 0.57 g cell /g MeOH , methanol consumption rate ([Formula: see text]) 5.76 g MeOH /h, and enzyme productivity ([Formula: see text]) 75.96 units/h. In conclusion, higher cell mass and β- glucosidase activity were produced under exponential feed rate than constant and linear feed rates., (© 2022. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

A recombinant form of pneumolysin from Streptococcus pneumoniae was obtained. By using Vector NTI Advance 11.0 bioinformatic analysis software, specific primers were designed in order to amplify the genome fragment of strain No. 3358 S. pneumoniae serotype 19F containing the nucleotide sequence encoding the full-length pneumolysin protein. A PCR product with a molecular weight corresponding to the nucleotide sequence of the S. pneumoniae genome fragment encoding the full-length pneumolysin was obtained. An expression system for recombinant pneumolysin in E. coli was constructed. Sequencing confirmed the identity of the inserted nucleotide sequence encoding the full-length recombinant pneumolysin synthesized in E. coli M15 strain. Purification of the recombinant protein was performed by affinity chromatography using Ni-Sepharose in 8 M urea buffer solution. Confirmation of the recombinant protein was performed by immunoblotting with monoclonal antibodies to pneumolysin., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Parvulins, peptidyl-prolyl isomerase enzymes (PPIase), catalyze the cis-trans isomerization of prolyl bonds in polypeptides, contributing to folding and function regulation of many proteins. Among Parvulins, Par17, exclusively expressed in hominids, is the least examined in terms of structure, catalytic function and cellular activity. Setting the conditions for the preparation of recombinant active Par17 may therefore significantly foster future studies. Here, we comparatively evaluated the impact of several parameters, including host strains, culture media, isopropyl ß-D-1-thiogalactopyranoside concentration, post-induction incubation time and temperature, on the overexpression of Par17 in E. coli cells. A similar approach was also comparatively adopted for the preparation of the recombinant full-length Pin1 protein, the most representative Parvulin, and the catalytic domains of both enzymes. Proteins were efficiently expressed and purified to homogeneity and were subjected to a structural characterization by Size Exclusion Chromatography and Circular Dichroism. Moreover, a single-step homogeneous protease-based fluorimetric assay, potentially scalable in HTS format, has been developed for determining the peptidyl-prolyl cis-trans isomerase activity of recombinant Parvulins. Results obtained show that proteins are folded and active. These new data mark an important milestone for progressing the investigation of Parvulins., (© 2022. The Author(s).)

Human Carnitine Acetyl Transferase (hCAT) reversibly catalyzes the transfer of the acetyl-moiety from acetyl-CoA to L-carnitine, modulating the acetyl-CoA/CoA ratio in mitochondria. Derangement of acetyl-CoA/CoA ratio leads to metabolic alterations that could result in the onset or worsening of pathological states. Due to the importance of CAT as a pharmacological target and to the European directive for reducing animal experimentation, we have pointed out a procedure to produce a recombinant, pure, and functional hCAT using the E. coli expression system. The cDNA encoding for the hCAT was cloned into the pH6EX3 vector. This construct was used to transform the E. coli Rosetta strain. The optimal conditions for the overexpression of the fully active hCAT include induction with a low concentration of IPTG (0.01 mM) and a low growth temperature (25 °C). The recombinant protein was purified from bacterial homogenate by affinity chromatography. The pure hCAT is very stable in an aqueous solution, retaining full activity for at least two months if stored at - 20 °C. These results could be helpful for a broad set of functional studies on hCAT, including drug-design applications., (© 2022. The Author(s).)