title: Purification of recombinant human epidermal growth factor secreted from the methylotrophic yeast Hansenula polymorpha.
abstract: The gene encoding human epidermal growth factor (hEGF) was expressed as a fusion protein with the Saccharomyces cerevisiae-derived prepro alpha-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 ysc(alpha) 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.
authors.affiliation: Biomolecular Process Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-600, Korea.
authors.forename: Joo-Hyung
authors.initials: JH
authors.lastname: Heo
authors.forename: Hye Soon
authors.initials: HS
authors.lastname: Won
authors.forename: Hyun Ah
authors.initials: HA
authors.lastname: Kang
authors.forename: Sang-Ki
authors.initials: SK
authors.lastname: Rhee
authors.forename: Bong Hyun
authors.initials: BH
authors.lastname: Chung
mesh_terms: D002268:Carboxypeptidases; D003001:Cloning, Molecular; D004815:Epidermal Growth Factor / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D002268:Carboxypeptidases; C078874:carboxypeptidase D