ABSTRACT
OBJECTIVES: Developing a Saccharomyces cerevisiae system for optimizing the expression of recombinant eukaryotic proteins. RESULTS: Two deletion mutants, which were hypersensitive to H2O2, were obtained by knocking out CTT1 and SOD2, respectively. The mutation rate of the mutants was up to over 4000 times of the spontaneous mutation rate when treated with H2O2. Endoglucanase Cel5A was used as a model enzyme to evaluate the system for improving the expression of the recombinant protein. Sixteen mutants of the RDKY3615 (ctt1∆) transformant and seven mutants of the RDKY3615 (sod2∆) transformant had significantly high Cel5A activity, while none mutants of the RDKY3615 transformant had significantly high enzyme activity. CONCLUSION: The combination of deletion mutagenesis and H2O2 treatment greatly accelerate the generation of genetic variants and will be a useful tool in improving the heterologous expression in S. cerevisiae.
Subject(s)
Catalase/metabolism , Cellulase/metabolism , Gene Deletion , Metabolic Engineering , Oxidative Stress , Saccharomyces cerevisiae/enzymology , Superoxide Dismutase/deficiency , Catalase/genetics , Hydrogen Peroxide/metabolism , Oxidants/metabolism , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/physiology , Superoxide Dismutase/geneticsABSTRACT
OBJECTIVE: To construct an immune alpaca phage display library, in order to obtain a single domain anti-BAFF (B cell-activating factor) antibody. METHODS: Using phage display technology, we constructed an immune alpaca phage display library, selected anti-BAFF single domain antibodies (sdAbs), cloned three anti-BAFF single-domain antibody genes into expression vector pSJF2, and expressed them efficiently in Escherichia coli. The affinity of different anti-BAFF sdAbs were measured by Bio layer interferometry. The in vitro biological function of three sdAbs was investigated by cell counting kit-8 (CCK-8) assay and a competitive enzyme-linked immunosorbent assay (ELISA). RESULTS: We obtained three anti-BAFF single domain antibodies (anti-BAFF64, anti-BAFF52 and anti-BAFFG3), which were produced in high yield in Escherichia coli and inhibited tumor cell proliferation in vitro. CONCLUSION: The selected anti-BAFF antibodies could be candidates for B-cell lymphoma therapies.
