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Development of expression systems for the production of recombinant human serum albumin using the MOX promoter in Hansenula polymorpha DL‐1
Author(s) -
Kang Hyun Ah,
Kang Whankoo,
Hong WonKyuong,
Kim Moo Woong,
Kim JeongYoon,
Sohn JungHoon,
Choi EuiSung,
Choe KeunBum,
Rhee Sang Ki
Publication year - 2001
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.1157
Subject(s) - recombinant dna , production (economics) , albumin , human serum albumin , biology , chemistry , microbiology and biotechnology , biochemistry , gene , economics , macroeconomics
To optimize the secretory expression of recombinant human serum albumin (HSA) under the control of methanol oxidase ( MOX ) promoter in the methylotrophic yeast Hansenula polymorpha DL‐1, we analyzed several parameters affecting the expression of HSA from the MOX promoter. Removal of the 5′‐untranslated region derived from HSA cDNA in the expression cassette led to at least a fivefold improvement of HSA expression efficiency at the translational level. With the optimized expression cassette, the gene dosage effect on HSA expression was abolished and thus, a single copy of the expression vector integrated into the MOX locus became sufficient for the maximal expression of HSA. Northern blot analysis revealed that the levels of HSA transcript did not increase any further upon increasing copy number. The mox ‐disrupted ( moxΔ ) transformant was constructed, in which the genomic MOX gene was transplaced with the HSA expression cassette, to examine the effect of the methanol oxidase‐deficient phenotype of the host on HSA expression. The moxΔ transformant showed higher levels of HSA production in shake‐flask cultures than the MOX wild‐type transformant, especially at low concentrations of methanol and a twofold higher specific HSA production rate in fed‐batch fermentation with an abrupt induction mode. The native prepro signal sequence of HSA secreted in H. polymorpha was correctly processed and the mature recombinant protein had a pI value identical to that of the authentic HSA. Our results suggest that the H. polymorpha expression systems developed in this study are suitable for large‐scale production of recombinant albumin. © John Wiley & Sons, Inc. Biotechnol Bioeng 76: 175–185, 2001.