Development of expression systems for the production of recombinant human serum albumin using the MOX promoter in Hansenula polymorpha DL-1

To optimize the secretory expression of recombinant human serum albumin (HS A) under the control of methanol oxidase (MOX promoter in the methylotrophi c yeast Hansenula polymorpha DL-1, we analyzed several parameters affecting the expression of HSA from the MOX promoter. Removal of the 5 ' -untransla ted region derived from HSA cDNA in the expression cassette led to at least a fivefold improvement of HSA expression efficiency at the translational l evel. 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 d id not increase any further upon increasing copy number. The mox-disrupted (mox Delta) 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 mo x Delta transformant showed higher levels of HSA production in shake-flask cultures than the MOX wild-type transformant, especially at low concentrati ons of methanol and a twofold higher specific HSA production rate in fed-ba tch fermentation with an abrupt induction mode. The native prepro signal se quence of HSA secreted in H. polymorpha was correctly processed and the mat ure recombinant protein had a pl value identical to that of the authentic H SA. Our results suggest that the H. polymorpha expression systems developed in this study are suitable for large-scale production of recombinant album in, (C) John Wiley & Sons, Inc.