期刊
PROCESS BIOCHEMISTRY
卷 38, 期 1, 页码 15-25出版社
ELSEVIER SCI LTD
DOI: 10.1016/S0032-9592(02)00037-7
关键词
IFN alpha-2a production; Hansenula polymorpha; co-expression of genes; protein processing; fermentation procedure; purification procedure
A DNA-sequence coding for IFNalpha-2a was expressed in the methylotrophic yeast Hansenula polymorpha from a strong inducible promoter element derived from the MOX gene, a key gene of the methanol metabolism pathway. For secretion the coding sequence was fused to the KEX2 recognition site of the S. cerevisiae-derived MFalpha1 prepro-leader. To a large extent the secreted molecules were found to be incorrectly processed from the precursor molecule exhibiting N-terminal extensions of the mature protein. Correct processing was achieved when co-expressing a S. cerevisiae-derived KEX2 gene from its native promoter. Undesirable proteolytic cleavage at additional dibasic sites of the protein sequence could be minimised when optimising fermentation conditions. A pH/pO(2)-controlled C-source feeding mode was applied to fermentations on a 1.5-10 l scale. In cultures of a transformant strain harbouring 30 copies of the IFN expression cassette a productivity of 350 mg/l could be obtained. Various capture procedures were found to be impaired when using a standard synthetic medium for culturing. Binding to ion exchange and hydrophobic interaction matrices was regained when using a modified YPG-based culture medium. (C) 2002 Elsevier Science Ltd. All rights reserved.
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