Search

Abstract Background Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. Results Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. Conclusion The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

β-Galactosidase enzymes continue to play an important role in food and pharmaceutical industries. These enzymes hydrolyze lactose in its constituent monosaccharides, glucose and galactose. The industrial use of enzymes presents an increase in process costs reflecting in higher final product value. An alternative to enhance processes' productivity and yield would be the use of recombinant enzymes and their large-scale fed-batch production. The overexpression of recombinant β-galactosidase from Kluyveromyces sp. was carried out in 2-L bioreactors using Escherichia coli strain BL21 (DE3) as host. Effect of induction time on recombinant enzyme expression was studied by adding 1 mM isopropyl thiogalactoside (IPTG) at 12 h, 18 h and 24 h of cultivation. Glucose feeding strategies were compared employing feedback-controlled DO-stat and ascendant linear pump feeding in bioreactor fed-batch cultivations. Linear feeding strategy with IPTG addition at 18 h of cultivation resulted in approximately 20 g/L and 17,745 U/L of biomass and β-galactosidase activity, respectively. On the other hand, although the feedback-controlled DO-stat feeding strategy induced at 12 h of cultivation led to lower final biomass of 18 g/L, it presented an approximately 2.5 increase in enzymatic activity, resulting in 42,367 U/L, and most importantly it led to the most prominent specific enzymatic activity of approximately 40 U/mgprotein. Comparing to previous results, these results suggest that the DO-stat feeding is a promising strategy for recombinant β-galactosidase enzyme production. [ABSTRACT FROM AUTHOR]

The aims of this work were to optimize the production of Erwinia carotovora l-asparaginase II enzyme in Escherichia coli by different fed-batch cultivation strategies using a benchtop bioreactor and to evaluate the therapeutic potential of the recombinant enzyme against different acute lymphoblastic leukemia cell lines. The highest enzyme activities (∼98,000 U/L) were obtained in cultures using the DO-stat feeding strategy with induction in 18 h of culture. Under these experimental conditions, the maximum values for recombinant l-asparaginase II (rASNase) yield per substrate, rASNase yield per biomass, and productivity were approximately 1204 U/g glucose , 3660 U/g cells , and 3260 U/(L·h), respectively. This condition was efficient for achieving high yields of the recombinant enzyme, which was purified and used in in vitro antileukemic potential tests. Of all the leukemic cell lines tested, RS4;11 showed the highest sensitivity to rASNase, with an IC 50 value of approximately 0.0006 U/mL and more than 70% apoptotic cells. The study demonstrated that the cultivation strategies used were efficient for obtaining high yield and productivity of rASNase with therapeutic potential inasmuch as cytotoxic activity and induction of apoptosis were demonstrated for this protein., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Hydrolysis efficiency of β-galactosidases is affected due to a strong inhibition by galactose, hampering the complete lactose hydrolysis. One alternative to reduce this inhibition is to perform mutations in the enzyme's active site. The aim of this study was to evaluate the effect of point mutations on the active site of different microbial β-galactosidases, using computational techniques. The enzymes of Aspergillus niger (AnβGal), Aspergillus oryzae (AoβGal), Bacillus circulans (BcβGal), Bifidobacterium bifidum (BbβGal), and Kluyveromyces lactis (KlβGal) were used. The mutations were carried out in all residues that were up to 4.5 Å from the galactose/lactose molecules and binding energy was computed. The mutants Tyr96Ala (AnβGal), Asn140Ala and Asn199Ala (AoβGal), Arg111Ala and Glu355Ala (BcβGal), Arg122Ala and Phe358Ala (BbβGal), Tyr523Ala, Phe620Ala, and Trp582Ala (KlβGal) had the best results, with higher effect on galactose binding energy and lower effect on lactose affinity. To maximize enzyme reactions by reducing galactose affinity, double mutations were proposed for BcβGal, BbβGal, and KlβGal. The double mutations in BcβGal and BbβGal caused the highest reduction in galactose affinity, while no satisfactory results were observed to KlβGal. Using computational tools, mutants that reduced galactose affinity without significantly affecting lactose binding were proposed. The mutations proposed can be used to reduce the negative feedback process, improving the catalytic characteristics of β-galactosidases and rendering them promising for industrial applications., (© 2020 American Institute of Chemical Engineers.)

Docking simulations have been used to assess protein complexes with some success. Small angle X-ray scattering (SAXS) is a well-established technique to investigate protein spatial configuration. This work describes the integration of geometric docking with SAXS to investigate the quaternary structure of recombinant human purine nucleoside phosphorylase (PNP). This enzyme catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for PNP causes gradual decrease in T-cell immunity. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant rejection, rheumatoid arthritis, lupus, and T-cell lymphomas. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. The present analysis confirms the trimeric structure observed in the crystal. The potential application of the present procedure to other systems is discussed.