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Title 

Rapid selection of multiple gene integrant for the production of recombinant hirudin in Hansenula polymorpha

Authors 

Hwa Young KimJung Hoon SohnChul Ho KimK Jagannadha RaoEui Sung ChoiMyung Kuk KimSang Ki Rhee

Publisher 

Springer Verlag (Germany)

Issue Date 

2000

Citation 

Biotechnology and Bioprocess Engineering, vol. 5, no. 1, pp. 1-6

Keywords 

hirudinHansenula polymorphaG418 resistancemulticopy integrationMOX promoter

Abstract 

For the rapid selection of higher recombinant hirudin producing strain in a methylotrophic yeast Hansenula 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 the MOX promoter of H. polymorpha and the mating factor α secretion signal of S. cerevisiae. Multiple integrants of a transforming 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 in H. 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.

ISSN 

1226-8372

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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