[Expression and secretion of human bone morphogenetic protein-7 in Pichia pastoris].
Sheng Wu Gong Cheng Xue Bao
; 22(6): 907-13, 2006 Nov.
Article
in Zh
| MEDLINE
| ID: mdl-17168311
ABSTRACT
The synonymous codons are used in a highly non-random manner in hosts of widely divergent species, which is termed "codon usage bias". Several reports suggest that codon usage bias sometimes frustrate attempts to express high levels of exogenous genes. In this study, we attempted to express mature peptide of human bone morphogenetic protein-7(hBMP7), with optimized codons in P. pastoris expression system. Three low-usage ARG codons (CGG or CGA) of gene fragment coding the mature peptide of hBMP7 have been successfully converted into P. pastoris-preferred ARG codons (AGA) by overlap extension PCR-based multiple-site-directed mutagenesis for a high level expression of hBMP7 mature peptide. The present results showed that the production level (25.45 mg/L) of codon-optimized hbmp7 had a remarkably improvement of 4.6-fold relative to that (5.5 mg/L) of non-codon-optimized hbmp7. Furthermore, a strain haboring multi-copy of codon-optimized hbmp7 expression cassette was screened, and showed a increased level of expression with 2-fold more potent than the single-copy one. The recombinant hBMP7 mature peptide were produced as a 18 kD monomer proteins, and were easily purified from culture supernatants by using ion-exchange chromatography. Functional assay demonstrated that rhBMP7 could induce ectopic cartilage formation, although its inductive ability was much less active than CHO cell-derived hBMP7.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Pichia
/
Bone Morphogenetic Protein 7
Limits:
Animals
/
Humans
Language:
Zh
Journal:
Sheng Wu Gong Cheng Xue Bao
Journal subject:
BIOTECNOLOGIA
Year:
2006
Document type:
Article
Affiliation country:
China