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Title 

Purification of recombinant human epidermal growth factor secreted from the methylotrophic yeast Hansenula polymorpha

Authors 

Joo Hyung HeoHye Soon WonHyun Ah KangSang Ki RheeBong Hyun Chung

Publisher 

Elsevier

Issue Date 

2002

Citation 

Protein Expression & Purification, vol. 24, no. 1, pp. 117-122

Keywords 

epidermal growth factorhumanisolation and purificationhumansrecombinant fusion proteins

Abstract 

The gene encoding human epidermal growth factor (hEGF) was expressed as a fusion protein with the Saccharomyces cerevisiae-derived prepro α-factor leader in the methylotrophic yeast Hansenula polymorpha. The recombinant hEGF(1-53), when secreted by H. polymorpha, rapidly cleaved to hEGF(1-52) by carboxy-terminal proteolysis, resulting in the accumulation of C-terminal-truncated hEGF(1-52) in the culture medium. To solve this problem, we constructed a H. polymorpha mutant in which the KEX1 gene coding for carboxypeptidase yscα was disrupted. The extent of C-terminal proteolysis of hEGF was significantly reduced when this kex1 disruptant was used as a host strain. After 24 h of shake-flask culture, most of the hEGF secreted by the kex1 disruptant remained intact, whereas more than 90% of the hEGF secreted by the wild-type was C-terminally cleaved. The recombinant hEGF was purified to >98% purity by two sequential steps of preparative scale anion exchange chromatography and reverse-phase HPLC. The authenticity of purified hEGF was confirmed by HPLC, N-terminal amino acid sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses.

ISSN 

1046-5928

Link 

http://dx.doi.org/10.1006/prep.2001.1527

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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