Enhanced production of human serum albumin by fed-batch culture of Hansenula polymorpha with high-purity oxygen.

Fed-batch cultures of Hansenula polymorpha were studied to develop an efficient biosystem to produce recombinant human serum albumin (HSA). To comply with this purpose, we used high purity oxygen supplying strategy to increase viable cell density in a bioreactor and enhance the production of target protein. A mutant strain, H. polymorpha GOT7 was utilized in this study as a host strain in both 5-L and 30-L scale fermentors. To supply high purity oxygen into a bioreactor, nearly 100 % high purity oxygen from commercial bomb or higher than 93 % oxygen available in-situ from a pressure swing adsorption oxygen generator (PSA) was employed. Under the optimal fermentation of H. polymorpha with high purity oxygen, the final cell densities and produced HSA concentrations were 24.6 g/L and 5.1 g/L in the 5-L fermentor, and 24.8 g/L and 4.5 g/L in the 30-L fermentor, respectively. These were about 2-10 times higher than those obtained in air-based fed-batch fermentations. The discrepancies between the 5-L and 30-L fermentors with air supply were presumably due to the higher contribution of surface aeration over submerged aeration in the 5-L fermentor. This study, therefore, proved the positive effect of high purity oxygen to enhance viable cell density as well as target recombinant protein production in microbial fermentations.

Maltose binding protein facilitates high-level expression and functional purification of the chemokines RANTES and SDF-1alpha from Escherichia coli.

The chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and SDF-1alpha (stromal cell-derived factor-1alpha) are important regulators of leukocyte trafficking and homing. Chemokines form insoluble inclusion bodies when expressed in Escherichia coli (E. coli), resulting in low yields of soluble protein. We have developed a novel chemokine expression system that generates a high amount of soluble protein and uses a simple purification scheme. We cloned different types of RANTES and SDF-1alpha fused to either maltose binding protein (MBP) or glutathione-S-transferase (GST) and expressed the fusion proteins in E. coli under various conditions. We found that the yield of soluble chemokine is influenced by the type of fusion partner. Fusion to MBP resulted in a higher yield of total and soluble chemokine compared to GST. Under optimized conditions, the yield of soluble MBP-RANTES and MBP-SDF-1alpha was 2.5- and 4.5-fold higher than that of the corresponding GST-fusion protein, respectively. Recombinant chemokine fusion proteins exhibited specific binding activity to chemokine receptors. These results demonstrate that the use of MBP-fusion proteins may provide an approach to generating high yields of soluble and functional chemokines, such as RANTES and SDF-1alpha.

Expression, purification, and antibody binding activity of human papillomavirus 16 L1 protein fused to maltose binding protein.

Genetic human papillomavirus type 16 L1 (HPV16 L1) has been widely studied for cervical cancer vaccine development. For the enzyme-linked immunosorbent assay (ELISA) screening of these vaccines, HPV16 L1 protein, which is required as a coating protein, has previously been expressed from costly and laborious recombinant baculovirus-infected insect cells. For a novel HPV16 L1 expression system characterized by a high yield of soluble form with simple purification steps, we have cloned and expressed two different types of HPV16 L1, both fused to maltose binding protein (MBP) or glutathione-S-transferase (GST) in Escherichia coli. The yield of soluble HPV16 L1 was influenced by the cultivation temperature. The yield of soluble form in the total MBP-fused HPV16 L1 protein (MBP-HPV16 L1) was 35% at 37 degrees C, but increased to 85% at 22 degrees C. Among the fusion partners, MBP provided higher yields of total and soluble HPV16 L1 than did GST. MBP-HPV16 L1 showed a 4.9-fold higher yield of the soluble form over insoluble inclusion bodies under optimized culture conditions. The soluble form of MBP-HPV16 L1 was purified via MBP affinity chromatography in a recovery yield of 9.7%. After fusion with MBP, HPV16 L1 showed binding activity to HPV16 L1-specific monoclonal antibody comparable to HPV16 L1 from the insect cells in ELISA tests. These results demonstrate that the use of MBP as a fusion partner may generate a high yield of soluble HPV16 L1 under optimized temperature conditions, and that MBP-fused HPV16 L1 might be applied further in evaluations of the immune responses of HPV16 L1-based cervical cancer vaccines.

Secretory expression and purification of Aspergillus niger glucose oxidase in Saccharomyces cerevisiae mutant deficient in PMR1 gene.

The gene encoding glucose oxidase (GOD) from Aspergillus niger was expressed as a secretory product in the yeast Saccharomyces cerevisiae. Six consecutive histidine residues were fused to the C-terminus of GOD to facilitate purification. The recombinant GOD-His(6) secreted by S. cerevisiae migrated as a broad diffuse band on SDS-PAGE, with an apparent molecular weight higher than that in natural A. niger GOD. To investigate the effects of hyperglycosylation on the secretion efficiency and enzyme properties, GOD-His(6) was expressed and secreted in a S. cerevisiae mutant in which the PMR1 gene encoding Ca(++)-ATPase was disrupted. The pmr1 null mutant strain secreted an amount of GOD-His(6) per unit cell mass higher than that in the wild-type strain. In contrast to the hyperglycosylated GOD-His(6) secreted in the wild-type strain, the pmr1 mutant strain secreted GOD-His(6) in a homogeneous form with a protein band pattern similar to that in natural A. niger GOD, based on SDS-PAGE. The hyperglycosylated and pmr1Delta mutant-derived GOD-His(6) enzymes were purified to homogeneity by immobilized metal ion-affinity chromatography and their specific activities and stabilities were compared. The specific activity of the pmr1Delta mutant-derived GOD-His(6) on a protein basis was very similar to that of the hyperglycosylated GOD-His(6), although its pH and thermal stabilities were lower than those of the hyperglycosylated GOD-His(6).