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

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Title
Purification of recombinant human epidermal growth factor secreted from the methylotrophic yeast Hansenula polymorpha
Author(s)
Joo Hyung Heo; Hye Soon Won; Hyun Ah Kang; Sang Ki Rhee; Bong Hyun Chung
Bibliographic Citation
Protein Expression and Purification, vol. 24, no. 1, pp. 117-122
Publication Year
2002
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
Publisher
Elsevier
DOI
http://dx.doi.org/10.1006/prep.2001.1527
Type
Article
Appears in Collections:
1. Journal Articles > Journal Articles
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