Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer
Autor: | John Walchli, Kaitlin Thompkins, Mark B. Mixon, Amy C. Raymond, Don Lorimer, Alex B. Burgin, Kimberly Archer, Ellen Wallace, Lance Stewart, Scott Lovell |
---|---|
Jazyk: | angličtina |
Rok vydání: | 2009 |
Předmět: |
lcsh:Biotechnology
Gene Expression Sequence alignment Engineered Gene Biology Protein Engineering 03 medical and health sciences chemistry.chemical_compound User-Computer Interface lcsh:TP248.13-248.65 Gene expression Escherichia coli Genes Synthetic Humans FtsZ Codon NS5B Gene 030304 developmental biology Genetics 0303 health sciences Base Composition Cell-Free System 030302 biochemistry & molecular biology Protein engineering Protein Structure Tertiary chemistry Codon usage bias biology.protein Sequence Alignment Algorithms Software Biotechnology Research Article |
Zdroj: | BMC Biotechnology BMC Biotechnology, Vol 9, Iss 1, p 37 (2009) |
ISSN: | 1472-6750 |
Popis: | Background With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. Results In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38α), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. Conclusion The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research. |
Databáze: | OpenAIRE |
Externí odkaz: |