Publication: Glutamic acid and alanine spacer is not necessary for removal of MFα-1 signal sequence fused to the human growth hormone produced from Pichia pastoris
Issued Date
2002-08-23
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ISSN
09593993
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2-s2.0-0036333438
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Mahidol University
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SCOPUS
Bibliographic Citation
World Journal of Microbiology and Biotechnology. Vol.18, No.6 (2002), 493-498
Suggested Citation
Lily Eurwilaichitr, Sittiruk Roytrakul, Chittiwat Suprasongsin, Pennapa Manitchotpisit, Sakol Panyim Glutamic acid and alanine spacer is not necessary for removal of MFα-1 signal sequence fused to the human growth hormone produced from Pichia pastoris. World Journal of Microbiology and Biotechnology. Vol.18, No.6 (2002), 493-498. doi:10.1023/A:1016383326627 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/19975
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Title
Glutamic acid and alanine spacer is not necessary for removal of MFα-1 signal sequence fused to the human growth hormone produced from Pichia pastoris
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Abstract
Human growth hormone (hGH) cDNA was synthesised using codons preferred by Escherichia coli, except for the first 20 amino acids, which were changed to that preferred by Saccharomyces cerevisiae and Pichia pastoris. Polymerase chain reaction (PCR) overlapping approach was employed to create synthetic hGH without glutamic acid-alanine (glu-ala), or with one and two glu-ala spacers (hGH, hGH1 and hGH2, respectively). The necessity of a glu-ala spacer in the cleavage of S. cerevisiae alpha mating factor-1 (MFα-1) secretion signal from the synthetic hGH was also investigated. Three types of hGH constructs were integrated into P. pastoris genome, the zeocin-resistant transformants were selected and expression of hGH was determined. A 22-kDa band of secreted hGH was further determined by N-terminal peptide sequencing. The result suggested that the removal of glu-ala from the hGH1 and hGH2 was not efficient and only the hGH construct showed the complete cleavage of the signal sequence, giving a similar N-terminus as the mature hGH. hGH expression was optimized to increase the yield of the protein from the hGH construct (no glu-ala) to 190 mg/l from a 10-ml induction medium.