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Large-Scale Preparation of Highly Stable Recombinant Human Acidic Fibroblast Growth Factor in Escherichia coli BL21(DE3) plysS Strain.

ABSTRACT: In this study, the optimum human aFGF gene encoding haFGF₁₃₅ was cloned in pET3c and transferred to Escherichia coli BL21(DE3) plysS. To enhance the yield of fermentation and the expression level of the target protein, the fermentation parameters, including temperature, pH, dissolved oxygen, glucose concentration, ammonium chloride concentration, induction time, and inducer (IPTG) concentration, were optimized. The optimized fermentation parameters were used in large-scale fermentation (30 L). Ion-exchange and heparin-affinity column chromatography techniques were used for separation and purification of rhaFGF₁₃₅ protein. HPLC, isoelectric focusing electrophoresis, and mass spectrometry were used to detect the purity, isoelectric point, and molecular weight and peptide map of rhaFGF₁₃₅ protein, respectively. Mitogenic activity of rhaFGF₁₃₅ protein was detected in NIH-3T3 cells and a full-thickness injury wound diabetic rat model. The production and expression level of rhaFGF₁₃₅ in the 30-L scale fermentation reached 80.4 ± 2.7 g/L culture and 37.8% ± 1.8%, respectively. The RP-HPLC and SDS-PAGE purity of the final rhaFGF₁₃₅ product almost reached 100%, and the final pure protein yield was 158.6 ± 6.8 mg/L culture. Finally, the cell and animal experiments showed that rhaFGF₁₃₅ retained a potent mitogenic activity. The large-scale process of rhaFGF₁₃₅ production reported herein is relatively stable and time-saving, and thus, it can be used as an efficient and economic strategy for the synthesis of rhaFGF₁₃₅ at the industrial level.

SUBMITTER: Yu B

PROVIDER: S-EPMC8072344 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The Escherichia coli B strain BL21(DE3) has had a profound impact on biotechnology through its use in the production of recombinant proteins. Little is understood, however, regarding the physiology of this important E. coli strain. We show here that BL21(DE3) totally lacks activity of the four [NiFe]-hydrogenases, the three molybdenum- and selenium-containing formate dehydrogenases and molybdenum-dependent nitrate reductase. Nevertheless, all of the structural genes necessary for the synthesis of the respective anaerobic metalloenzymes are present in the genome. However, the genes encoding the high-affinity molybdate transport system and the molybdenum-responsive transcriptional regulator ModE are absent from the genome. Moreover, BL21(DE3) has a nonsense mutation in the gene encoding the global oxygen-responsive transcriptional regulator FNR. The activities of the two hydrogen-oxidizing hydrogenases, therefore, could be restored to BL21(DE3) by supplementing the growth medium with high concentrations of Ni²⁺ (Ni²⁺-transport is FNR-dependent) or by introducing a wild-type copy of the fnr gene. Only combined addition of plasmid-encoded fnr and high concentrations of MoO₄²⁻ ions could restore hydrogen production to BL21(DE3); however, to only 25-30% of a K-12 wildtype. We could show that limited hydrogen production from the enzyme complex responsible for formate-dependent hydrogen evolution was due solely to reduced activity of the formate dehydrogenase (FDH-H), not the hydrogenase component. The activity of the FNR-dependent formate dehydrogenase, FDH-N, could not be restored, even when the fnr gene and MoO₄²⁻ were supplied; however, nitrate reductase activity could be recovered by combined addition of MoO₄²⁻ and the fnr gene. This suggested that a further component specific for biosynthesis or activity of formate dehydrogenases H and N was missing. Re-introduction of the gene encoding ModE could only partially restore the activities of both enzymes. Taken together these results demonstrate that BL21(DE3) has major defects in anaerobic metabolism, metal ion transport and metalloprotein biosynthesis.

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Large-Scale Preparation of Highly Stable Recombinant Human Acidic Fibroblast Growth Factor in Escherichia coli BL21(DE3) plysS Strain.

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