BMB Reports
- Volume 34 Issue 6
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- Pages.502-508
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- 2001
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- 1976-670X(eISSN)
A Rapid and Simple Method for Construction and Expression of a Synthetic Human Growth Hormone Gene in Escherichia coli
- Roytrakul, Sittiruk (National Center for Genetic Engineering and Biotechnology (BIOTEC)) ;
- Eurwilaichitr, Lily (National Center for Genetic Engineering and Biotechnology (BIOTEC)) ;
- Suprasongsin, Chittiwat (Ramathibodi Hospital) ;
- Panyim, Sakol (BIOTEC Training Center for Genetic Engineering and Biotechnology at Institute of Molecular Biology and Genetics, Mahidol University)
- Received : 2001.05.08
- Accepted : 2001.07.31
- Published : 2001.11.30
Abstract
A cDNA, encoding the human growth hormone (hGH), was synthesized based on the known 191 amino acid sequence. Its codon usage was optimized for a high level expression in Escherichia coli. Unique restriction sites were incorporated throughout the gene to facilitate mutagenesis in further studies. To minimize an initiation translation problem, a 624-bp cassette that contained a ribosome binding site and a start codon were fused to the hGH-coding sequence that was flanked between the EcoRI and HindIII sites. The whole fragment was synthesized by an overlapped extension of eight long synthetic oligonucleotides. The four-short duplexes of DNA, which were first formed by annealing and filling-in with a Klenow fragment, were assembled to form a complete hGH gene. The hGH was cloned and expressed successfully using a pET17b plasmid that contained the T7 promoter. Recombinant hGH yielded as much as 20% of the total cellular proteins. However, the majority of the protein was in the form of insoluble inclusion bodies. N-terminal amino acid sequencing also showed that the hGH produced in E. coli contained formyl-methionine. This study provides a useful model for synthesis of the gene of interest and production of recombinant proteins in E. coli.