1. Effect of Clp protease from Corynebacterium glutamicum on heterologous protein expression.
- Author
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Liu X, Meng L, Wang X, Yang Y, and Bai Z
- Subjects
- Computational Biology methods, Corynebacterium glutamicum enzymology, Endopeptidase Clp deficiency, Fermentation, Gene Knockout Techniques, Genes, Reporter, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Humans, Immunoglobulin Heavy Chains genetics, Immunoglobulin Heavy Chains isolation & purification, Isoenzymes deficiency, Isoenzymes genetics, Recombinant Proteins biosynthesis, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Teriparatide isolation & purification, Transgenes, Corynebacterium glutamicum genetics, Endopeptidase Clp genetics, Gene Expression Regulation, Bacterial, Immunoglobulin Heavy Chains biosynthesis, Teriparatide metabolism
- Abstract
The protease present in a host may reduce the yield and biological activity of heterologous proteins. In this study, we used protease overexpression and deletion strategies to examine the effect of the Clp protease system in Corynebacterium glutamicum on the recombinant protein and to produce a highly efficient heterologous protein expression host. In this study, we identified seven genes in the Clp protease family in Corynebacterium glutamicum ATCC 13032 through bioinformatics analysis, and studied their effects on the enhanced green fluorescent protein (EGFP) reporter protein. The fluorescence intensity of the knockout strain was significantly higher, and the effect of the clpS deletion strain was the most obvious. To verify the universal effect of the lack of clpS, the excellent industrial strain C. glutamicum 1.15647 was transformed to form recombinant 15647-ΔclpS. Based on the results, 15647-ΔclpS had a more significant effect on improving protein expression. Furthermore, recombinant human teriparatide (rhPTH) and variable domain of heavy chain of heavy-chain antibody (VHH) were selected to verify the universal applicability of the knockout strain for expressing heterologous proteins. Accordingly, we found that protease deficiency could increase the production of heterologous proteins. Finally, through a large-scale fermentation, the 15647-ΔclpS strain was used to produce VHH. Its yield was approximately 530 mg/L, which was 65% higher than that of WT-15647. In this study, a host that could effectively increase heterologous protein expression was successfully obtained., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2022
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