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KMID : 1094720000050010001
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Biotechnology and Bioprocess Engineering
2000 Volume.5 No. 1 p.1 ~ p.6
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Rapid selection of multiple gene integrant for the production of recombinant hirudin inHansenula polymorpha
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Kim Hwa-Young
Sohn Jung-Hoon
Kim Chul-Ho
Rao K. Jagannadha
Choi Eui-Sung
Kim Myung-Kuk
Rhee Sang-Ki
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Abstract
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For the rapid selection of higher recombinant hirudin producing strain in a methylotrophic yeastHansenula polymorpha, a multiple gene integration and dose-dependent selection vector, based on a telomere-associated ARS and a bacterial aminoglycoside 3-phosphotransferase ( (aph) gene, was adopted. Two hirudin expression cassettes (HV1 and HV2) were constructed using theMOX promoter ofH. polymorpha and the mating factor α secretion signal ofS. cerevisiae. Multiple integrants of a transformang vector containing hirudin expression cassettes were easily selected by using an antibiotic, G418. Hirudin expression level and integrated plasmid copy number of the tested transformants increased with increasing the concentration of G418 used for selection. The expression level of HV1 was consistently higher than that of HV2 under the similar conditions, suggesting that the gene context might be quite important for the high-level gene expression inH. polymorpha. The highest hirudin producing strain selected in this study produced over 96 mg/L of biologically active hirudin in a 500-mL flask and 165 mg/L in a 5-L fermentor.
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KEYWORD
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hirudin, Hansenula polymorpha, G418 resistance, multicopy integration, MOX promoter
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