Expression and characterization of recombinant human antithrombin III in Pichia pastoris.

Antithrombin III (ATIII) is a member of the serpin superfamily and a major regulator of the blood coagulation cascade. To express recombinant human ATIII (rATIII) in the methylotrophic yeast Pichia pastoris, we constructed an rATIII expression plasmid which contained the ATIII cDNA encoding mature protein region connected with the truncated mAOX2 promoter and the SUC2 secretion signal, introduced it into the P. pastoris genome, and screened for a single copy transformant. The secretion of rATIII from the transformant reached a level of 320 IU/L in the culture broth at 169 h. From the culture-supernatant, rATIII was purified to over 99% by heparin-affinity chromatography and other column chromatography methods. We characterized rATIII and compared it with human plasma-derived ATIII (pATIII). The purified rATIII possessed correct N-terminal amino acid sequence, and its molecular weight by SDS-PAGE of 56,000 Da was slightly different from the 58,000 Da of pATIII. Sequence and mass spectrometry analysis of BrCN fragments revealed that posttranslational modifications had occurred in rATIII. O-linked mannosylation was found at Ser 3 and Thr 9, and in some rATIII molecules, modification with O-linked mannosyl-mannose had probably occurred at Thr 386, close to the reactive center. Although the heparin-binding affinity of rATIII was 10-fold higher than that of pATIII, its inhibitory activity against thrombin was only half. As the conformation of rATIII and pATIII by circular dichroism spectroscopy was similar, O-glycosylation in the reactive center loop was assumed to be mainly responsible for the decreased inhibitory activity. pATIII can inactivate thrombin through formation of a stable thrombin-ATIII complex, but rATIII modified with O-glycosylation in the reactive center loop may act as a substrate rather than an inhibitor of thrombin.

High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation.

The methylotrophic yeast, Pichia pastoris, is widely used as a host strain for the production of a variety of heterologous proteins. We used P. pastoris for the production of recombinant human serum albumin (rHSA). In several runs of fed-batch fermentation, rapid degradation of rHSA was observed, coinciding with a sudden increase of protease activity in the culture broth. Monitoring the changes in the concentration of the medium components during fermentation suggested that this phenomenon was caused by nitrogen starvation. Increased initial concentrations of ammonia and phosphoric acid in the medium prevented the protease production during fermentation. Using this improved medium, stable production of rHSA of around 1.4 g/l was achieved. Although protease activity in the culture broth of the improved medium was not detected by the casein plate method at the end of fermentation, potential protease activity remained and could be activated by decreasing the pH of the culture broth, a high degradation rate of 660 mg HSA/l/h was observed at pH 4.3, but degradation did not occur above pH 5.9.

Addition of oleic acid increases expression of recombinant human serum albumin by the AOX2 promoter in Pichia pastoris.

The addition of several kinds of fatty acid to the culture medium of a recombinant human serum albumin (rHSA)-producing yeast, Pichia pastoris, resulted in increased expression levels of the product. Among the fatty acids tested, a small amount of oleic acid (0.01% (w/v)) doubled the rHSA production level in a shake-flask culture when measured by the reversed passive hemagglutination assay method. To elucidate this phenomenon, studies were conducted using deletion mutants from the AOX2 promoter region. Deletion mutants, designed for a detailed evaluation of the methanol regulation elements (AOX2-UAS, AOX2-URS1, and AOX2-URS2) did not respond to the addition of oleic acid. However, a deletion mutant that was not lacking an upstream region from the AOX2 promoter showed a response to oleic acid. The results implied the presence of an oleic acid-responsive element between nucleotides (nt) -1529 and -803, and it may lie between nt -1411 and -1403 in the AOX2 promoter of P. pastoris. The response to oleic acid was shown to function even when the level of rHSA expression was increased by a mutation in the AOX2 promoter. Therefore addition of oleic acid to the medium is likely to play an important role, in cooperation with gene manipulation, in achieving high expression levels of rHSA for the purpose of commercial production.

High level secretion of recombinant human serum albumin by fed-batch fermentation of the methylotrophic yeast, Pichia pastoris, based on optimal methanol feeding strategy.

The dynamic programming method was applied to obtain the optimal specific growth rate, mu, in the fed-batch fermentation using the recombinant human serum albumin (rHSA)-producing yeast, Pichia pastoris. Based on the relationship between the specific production rate, varrho, and the specific growth rate, mu, a simple mathematical model describing the growth and rHSA production was constructed and used for calculations. Two constraints, final volume and maximum methanol feed rate, were adopted for calculations and the optimal mu resulted as follows. That is, mu was initially at the maximum value, mu(max), then decreased gradually. Finally, mu decreased to the mu(min) that gave a maximum varrho. The decline of mu was revealed to be caused by the constraint for maximum methanol feeding rate, F(max), and F(max) was constant until mu decreased to mu(min). We tried to realize the optimal mu in the fed-batch fermentation by manipulating the methanol feeding rate and obtained it. However, the observed varrho was differed from the expected one. The discrepancy between the expected varrho and observed varrho after the change of mu suggests the inapplicability of the relationship between mu and varrho to dynamic situations where mu changes. To confirm this, simulation and fed-batch fermentation runs were carried out at a methanol feeding rate that would cause a continuous change in mu. The rHSA production was simulated well, suggesting the applicability of the relationship between mu and varrho in such situations. Discontinuity in the change in methanol feeding rate of the optimal feed pattern at the time mu changed is considered to be the cause for the discrepancy between the expected and observed varrho. Therefore, a new methanol feeding strategy that could mimic the changes in mu and varrho of the optimal strategy without a discontinuity in the feeding rate was sought using a mathematical model of fermentation by trial and error. This modification in the methanol feeding rate resulted in a considerably improved varrho and 18% increase in total rHSA production compared with those obtained by the optimal strategy.