Expression and secretion of human bone morphogenetic protein-7 in pichia pastoris |
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Authors: | Ling CHEN Yi-De HUANG Yan-Ding ZHANG |
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Affiliation: | aCollege of Life Sciences, Fujian Normal University, Fuzhou 350007, China;bState Key Laboratory of Developmental Biology and Neurobiology, Fujian Normal University, Fuzhou 350007, China;cKey Laboratory of Sugarcane Genetics and Breeding, Ministry of Agriculture, Fuzhou 350007, China |
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Abstract: | The synonymous codons are used in a highly nonrandom manner in hosts of widely divergent species, which is termed ‘codon usage bias’. Several reports suggest that codon usage bias sometimes obstructs attempts to express high levels of exogenous genes. In this study, an attempt was made to express mature peptide of human bone morphogenetic protein-7 with optimized codons in P. pastoris expression system. Three low-usage ARG codons (CGG or CGA) in hBmp7 mature domain have been successfully transformed into P. pastoris-preferred ARG codons (AGA) with overlap extension PCR-based multiple-site-directed mutagenesis for a high level expression of hBMP7 mature peptide. The results of this study showed that the production level (25.45 mg/L) of a codon-optimized strain increased 4.6-fold in comparison with that (5.5 mg/L) of noncodon-optimized strain. A strain harboring multicopy of codon-optimized hbmp7 expression cassette showed an even higher expression level, which was about 2-fold compared with that of the single-copy one. These recombinant hBMP7 mature peptides were produced as 18-kD monomer proteins and were easily purified from culture supernatants using ion-exchange chromatography. Functional assay demonstrated that rhBMP7 could induce ectopic cartilage formation, although its inductive ability was much less active than that of CHO cell-derived hBMP7. |
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Keywords: | hBMP7 Condon usage bias site-directed mutagenesis Pichia pastoris |
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