Sittiruk RoytrakulLily EurwilaichitrChittiwat SuprasongsinSakol PanyimThailand National Center for Genetic Engineering and BiotechnologyMahidol UniversityFaculty of Medicine, Ramathibodi Hospital, Mahidol University2018-09-072018-09-072001-11-30Journal of Biochemistry and Molecular Biology. Vol.34, No.6 (2001), 502-508122586872-s2.0-0035601508https://repository.li.mahidol.ac.th/handle/20.500.14594/26435A 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.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyArticleSCOPUS