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Background The methylotrophic yeast Pichia pastoris is an appealing production host for a variety of recombinant proteins, including biologics. In this sense, various genetic- and non-genetic-based techniques have been implemented to improve the production efficiency of this expression platform. Loss of supression (Los1) encodes a non-essential nuclear tRNA exporter in Saccharomyces cerevisiae, which its deletion extends replicative lifespan. Herein, a los1-deficient strain of P. pastoris was generated and characterized. Methods A gene disruption cassette was prepared and transformed into an anti-CD22-expressing strain of P. pastoris. A δ los1 mutant was isolated and confirmed. The drug sensitivity of the mutant was also assessed. The growth pattern and the level of anti-CD22 single-chain variable fragment (scFv) expression were compared between the parent and mutant strains. Resuults The los1 homologue was found to be a non-essential gene in P. pastoris. Furthermore, the susceptibility of los1 deletion strain to protein synthesis inhibitors was altered. This strain showed an approximately 1.85-fold increase in the extracellular level of anti-CD22 scFv (p < 0.05). The maximum concentrations of total proteins secreted by δ los1 and parent strains were 125 mg/L and 68 mg/L, respectively. Conclusion The presented data suggest that the targeted disruption of los1 homologue in P. pastoris can result in a higher expression level of our target protein. Findings of this study may improve the current strategies used in optimizing the productivity of recombinant P. pastoris strains.

Superficial fungal infections are prevalent worldwide, with dermatophytes, as the most common cause. Various antifungal agents including azoles and allylamines are commonly used to treat dermatophytosis. However, their overuse has yielded drug-resistant strains, calling for the development of novel anti-mycotic compounds. Olorofim, is a newly developed antifungal compound, which targets pyrimidine biosynthesis in molds. The purpose of this study was to determine the in vitro and in vivo antifungal effects of olorofim against common dermatophytes. The in vitro activity of olorofim against dermatophytes was assessed by microtiter broth dilution method. Bioinformatic analysis of olorofim binding to dihydroorotate dehydrogenase (DHODH) of dermatophytes was also performed, using Aspergillus fumigatus DHODH as a template. The in vivo efficacy of the drug was investigated, using a guinea pig model, experimentally infected with Microsporum gypseum. Microtiter assays confirmed the high in vitro sensitivity of dermatophytes to olorofim (MIC= 0.015-0.06 mg/L). Amino acid sequence analysis indicated that DHODH is highly conserved among dermatophytes. The critical residues, in dermatophytes, involved in olorofim binding, were similar to their counterparts in A. fumigatus DHODH, which explains their susceptibility to olorofim. Typical skin lesions of dermatophyte infection, were observed in the guinea pig model, at seven days post-inoculation. Following one week of daily topical administration of olorofim, similar to the clotrimazole group, the skin lesions were resolved and normal hair growth patterns appeared. In light of the in vitro and in vivo activity of olorofim against dermatophytes, this novel agent may be considered as a treatment of choice, against dermatophytosis.

Multifunctional matrix protein (M) of rabies virus (RABV) plays essential roles in the pathogenesis of rabies infection. Identification of M protein interacting partners in target hosts could help to elucidate the biological pathways and molecular mechanisms involved in the pathogenesis of this virus. In this study, two-dimensional Far-western blotting (2D-Far-WB) technique was applied to find possible matrix protein partners in the rat brainstem. Recombinant RABV M was expressed in Pichia pastoris and was partially purified. Subsequently, 2D-Far-WB-determined six rat brainstem proteins interacted with recombinant M proteins that were identified by mass spectrometry. Functional annotation by gene ontology analysis determined these proteins were involved in the regulation of synaptic transmission processes, metabolic process and cell morphogenesis–cytoskeleton organization. The interaction of viral M protein with selected host proteins in mouse Neuro-2a cells infected with RABV was verified by super-resolution confocal microscopy. Molecular docking simulations also demonstrated the formation of RABV M complexes. However, further confirmation with co-immunoprecipitation was only successful for M-actin cytoplasmic 1 interaction. Our study revealed actin cytoplasmic 1 as a binding partner of M protein, which might have important role(s) in rabies pathogenesis.

Aims The Escherichia coli expression system is highly effective in producing recombinant proteins. However, there are some limitations in this system, especially in obtaining correctly folded forms of some complex proteins such as Fab fragments. To improve the solubility and folding quality of Fab fragments, we have examined the effect of simultaneous application of a SUMO fusion tag, EnBase® cultivation mode and a redox mutant strain in the E. coli expression system. Methods and results A bicistronic gene construct was designed to express an antivascular endothelial growth factor (VEGF) Fab fragment as a model system. The construct contained a dual SUMO fusion gene fragment to encode SUMO-tagged heavy and light chains. While the expression of the construct in batch cultures of BL21 or SHuffle® transformants produced insoluble and unfolded products, the induction of the transformants in EnBase® medium resulted in soluble and correctly folded Fab fragment, reaching as high as 19% of the total protein in shuffle strain. The functional assays indicated that the biological activity of the target Fab is similar to the commercial anti-VEGF, Lucentis® . Conclusions This study demonstrated that the combination of SUMO fusion technology, EnBase® cultivation system and recruiting a redox mutant of E. coli can efficiently enhance the solubility and productivity of recombinant Fab fragments. Significance and the impact of the study The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments.

In Saccharomyces cerevisiae, los1 encodes a nuclear tRNA exporter. Despite the non-essentiality, the deletion of los1 has been shown to extend replicative life span in yeast. Here, we characterized AfuXpot, the los1 homologue in human pathogen Aspergillus fumigatus and found that it is continuously expressed during fungal growth. Microscopic examination of an AfuXpot-GFP-expressing transformant confirmed the nuclear localization of the fusion protein. The targeted gene deletion affirmed the non-essential role of AfuXpot in hyphal growth and sporulation. However, the growth of the deletion mutant was affected by amino acid, but not glucose, deprivation. The susceptibility of the deletant strain to protein and DNA/RNA synthesis inhibitors was also altered. Using bioinformatics tools, some transcription factor binding sites were predicted in AfuXpot promoter. Expression analyses of potential AfuXpot-interacting genes showed a marked down-regulation of sfp1 and mtr10 homologues in ΔAfuXpot strain. Our data demonstrates some conserved aspects of AfuXpot as a tRNA exporter in A. fumigatus.

An RNAi system based on T7 RNA polymerase (TRNAP) was designed and examined in Aspergillus fumigatus. This system consists of two elements; an inducible T7RNAP expressing cassette and an AMA1-based episomal RNAi plasmid. These constructs were transformed into the A. fumigatus protoplasts and the efficiency of this system was tested in downregulation of alb1 gene. Upon the induction of T7RNAP expression, the recombinant T7RNAP was able to recognize T7 promoters, which were located on the episomal plasmid and in opposite direction. As a result, the bidirectional transcription of alb1 fragment led to the silencing of the target gene. However, our results demonstrated that this silencing system is unstable and may not be applicable in preparation of RNAi libraries.

International audience; In Saccharomyces cerevisiae, Nce102 encodes a 173 amino acid transmembrane protein, which acts as a key player in eisosome assembly and plasma membrane organization. Here, we describe the characterization of Nce102 homologue in the human pathogen, Aspergillus fumigatus. Our results demonstrated that AfuNce102 is continuously expressed during fungal growth. In addition, microscopic examination of an AfuNce102-GFP-expressing transformant confirmed the localization of the fusion protein to the endoplasmic reticulum with higher density fluorescence at the tip of the mycelium. During conidiogenesis, the protein was localized to the conidiophores and the conidia. Abnormal conidiation of AfuNce102 deletion mutant suggests a potential role for AfuNce102 in sporulation process.

Objective: The production of heterologous proteins in Escherichia coli is strongly affected by codon bias. This phenomenon occurs when the codon usage of mRNA coding for the foreign protein differs from that of the bacterium. The ribosome pauses upon encountering a rare codon and may detach from mRNA, thereby the yield of recombinant protein production reduces. The aim of this study is to investigate the effect of these codon numbers reductions on the recombinant protein production. Materials and Methods: Since most amino acids are encoded by more than one codon, codons were changed in order to their usage in a special host such as E. coli without any transformation in amino acids sequence. Silent mutations in 5' codons of human basic fibroblast growth factor cDNA carried out by site-directed mutagenesis and the expression level of the recombinant protein is analyzed by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Results: Expression level in mutant and wild-type genes indicated a considerable difference. In contrast with the remarkable bands of wild-type gene in all the strains particularly in codon plus strain, there were no significant bands related to mutant gene in SDS-PAGE analysis. Conclusion: Because of the same conditions of mutant and wild-type genes during the translation and transcription, this significant difference may relate to mRNA efficiency for translation. Our results indicate that increased stability of 5' mRNA secondary structures in E. coli prevents efficient translation initiation. Furthermore, wild-type gene significant bands in codon plus strain support the hypothesis that the possible elimination of translational pauses that increase translation rate leads to over expression.

International audience; Annexin C4 has been identified as a new member of fungal annexin family. In search of function, we have generated an annexin C4 disruptant strain of human pathogen, Aspergillus fumigatus. Detailed phenotypic analysis confirmed a non essential role of annexin C4 in the growth and sporulation of this pathogen. We applied a comparative proteomics strategy to understand the possible role of this protein in the fungus. The modification of respiratory chain proteins and stress response proteins suggests the occurrence of a mild oxidative stress in anxC4 disruptant strain. This may indicate a possible anti stress function of annexin C4 in A. fumigatus.

Urate oxidase is an important enzyme with therapeutic and diagnostic applications. Rasburicase is a recombinant urate oxidase enzyme approved by FDA to use in the treatment of hyperuricemia conditions. Various hosts such as Saccharomyces cerevisiae, Hansenula polymorpha and Escherichia coli have been used to express the enzyme. Today, Pichia pastoris is considered as an important host for heterologous protein expression since it has beneficial characteristics such as strong promoters, simple scale up, post translational modifications, high cell density cultivation and simple genetic manipulation. In this study, Aspergillus flavus urate oxidase gene was cloned in pPICZαA expression vector and expressed in P. pastoris. The recombinant urate oxidase was expressed in secretory form and was confirmed through RT-PCR, SDS-PAGE analysis and western blotting. The enzyme activity was determined using a colorimetric assay. A production yield of 0.43 U/ml of culture supernatant was obtained.

International audience; AIMS: A novel chimeric-truncated form of t-PA with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in E.coli (BL21) strain and compare the protein potency in batch and fed-batch processes. METHODS AND RESULTS: The expression cassette for the novel t-PA was prepared in pET-28a(+). The E.coli expression procedure was compared in traditional batch and newly developed fed batch; EnBase® Flo system. The protein was purified in soluble format and potency results were identified using Chromolize t-PA assay kit. The fed-batch fermentation mode, coupled with a Ni-NTA affinity purification procedure under native condition resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46.66 IU mg(-1) ) compared to traditional batch mode (35.8 IU mg(-1) ). CONCLUSIONS: Considering the undeniable advantages of expression in the prokaryotic expression systems such as E.coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed-batch cultivation methods showed the potential to replace miss folded formats of protein with proper folded, soluble form with improved potency. SIGNIFICANCE AND IMPACT OF STUDY: E.coli expression of recombinant proteins, still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system. © 2012The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.