Expression of glycoproteins bearing complex human-like glycans with galactose terminal in Hansenula polymorpha.

Glycoproteins derived from Hansenula polymorpha can not be used for therapeutic purposes due to their high-mannose type asparagine-linked (N-linked) glycans, which result in immune reactions and poor pharmacokinetic behaviors in human body. Previously, we reported that the trimannosyl core N-linked glycans (Man(3)GlcNAc(2)) intermediate can be generated in endoplasmic reticulum in HpALG3 and HpALG11 double-mutant H. polymorpha. Here, we describe the further modification of the glycosylation pathway in this double-defect strain to express glycoproteins with complex human-like glycans. After eliminating the impact of HpOCH1, three glycosyltransferases were introduced into this triple-mutant strain. When human ß-1,2-N-acetylglucosaminyltransferase I (hGnTI) was efficiently targeted in early Golgi, more than 95 % glycans attached to the glycoproteins were added one N-acetylglucosamine (GlcNAc). With subsequently introduction of rat ß-1,2-N-acetylglucosaminyltransferase II (rGnTII) and human ß-1,4-galactosyltransferase I (hGalTI), several glycoengineered strains can produce glycoproteins bearing glycans with terminal N-acetylglucosamine or galactose. The expression of glycoproteins with glycan Gal(2)GlcNAc(2)Man(3)GlcNAc(2) represents a significant step toward the ability to express fully humanized glycoproteins in H. polymorpha. Furthermore, several shake-flask and bioreactor fermentation experiments indicated that, although the cells do display a reduction in growth rate, the glycoengineered strains are still suitable for high-density fermentation.

Hansenula polymorpha expressed heat shock protein gp96 exerts potent T cell activation activity as an adjuvant.

Previous studies together with ours showed that heat shock protein gp96 as an adjuvant induces antigen specific T cell responses against cancer and infectious diseases. However, at present there is no efficient method to obtain high amount of full-length gp96 by in vitro expression. Here, we used the yeast Hansenula polymorpha as an efficient host for gp96 recombinant protein production. The transformant clones with highly expressed recombinant proteins were screened and selected by measuring the halo size which indicates enzymatic hydrolysis of starch in the medium. High-level production of gp96 (around 150mg/mL) was achieved by using high-cell density fed-batch cultivations. We showed that peptide binding of the recombinant protein has similar specificity and intrinsic binding parameters as that of the native gp96. We next examined the self-assembly properties and high-order structures of the recombinant protein. Moreover, the H. polymorpha expressed recombinant gp96 can effectively induce HBV-specific CTL response in immunized mice while Escherichia coli-expressed gp96 cannot. Our results therefore may provide bases for structure and functional studies of gp96 and thereby potentially expedite the development of gp96-based vaccines for immunotherapy of cancer or infectious diseases.

Identification and functional characterization of the HpALG11 and the HpRFT1 genes involved in N-linked glycosylation in the methylotrophic yeast Hansenula polymorpha.

The initial steps in N-linked glycosylation involve the synthesis of a lipid-linked core oligosaccharide followed by the transfer of the core glycan to nascent polypeptides in the endoplasmic reticulum (ER). In this study, we have identified two genes, HpALG11and HpRFT1, in the metylotrophic yeast Hansenula polymorpha. Detailed analysis of the glycan structures of the N-linked glycans of secreted recombinant glucose oxidase in mutant strains Hpalg3Δ, Hpalg11Δ, and Hpalg3Δalg11Δ with the assistance of over-expression of RFT1 was performed by linkage-specific mannosidase digestion. The results suggest that HpALG11 and HpRFT1 were responsible for catalyzing the sequential transfer of terminal α-1,2-Man residues to form the Man(5)GlcNAc(2)-PP-Dol intermediate at the cytosolic side of the ER before flipping to the luminal side and encoding an evolutionarily conserved protein required for the translocation of Man(5)GlcNAc(2)-PP-Dol from the cytoplasmic to the lumenal leaflet of the ER membrane, respectively. Deletion of the HpALG11 gene leads to poor growth and temperature-sensitive lethality, whereas over-expression of HpRft1p can improve growth of the Hpalg11Δ and Hpalg3Δalg11Δ strains. Furthermore, deletion of the HpALG11 gene in the Hpalg3Δ strain resulted in the secretion of glycoproteins with a predicted structure mainly containing trimannosyl core N-linked glycans (Man(3)GlcNAc(2)).

Cloning and expression of Aspergillus tamarii FS132 lipase gene in Pichia pastoris.

A lipase gene (atl) was cloned from Aspergillus tamarii FS132 for the first time. The gene was found to have an open reading frame of 1024 base pairs (bp), and the coding region of the gene contained two introns (51 bp and 52 bp). Multi-alignment analysis of the deduced amino acid sequence indicated high homology between the enzyme and mono-and diacylglycerol lipases from fungi Aspergillus. The recombinant lipase was expressed in Pichia pastoris GS115 cells. The recombinant lipase was found to have a molecular mass of 36.7 kDa, and it exhibited lipase activity of 20 U/mL in culture supernatant when tributyrin was used as the substrate.

Development of an α-amylase reporter system for efficient screening of clones with highly expressed heterologous protein in Hansenula polymorpha.

To check feasibility and effectiveness of the α-amylase reporter system, two vectors were designed and tested using hepatitis B virus surface antigen (HBsAg) and Homo sapiens granulocyte-macrophage colony stimulating factor 2 (hGM-CSF2) as a model. By integrating the vector containing two independent cassettes into the same genome locus, high-producing clones of HBsAg (or hGM-CSF2) were screened using the α-amylase as a reporter. Results show there was a positive correlation (Correlation coefficient, R (2) > 0.95) between the yield of recombinant proteins and the α-amylase activity of corresponding transformants, which was independent of the gene dosage.

Improved gene disruption method and Cre-loxP mutant system for multiple gene disruptions in Hansenula polymorpha.

In H. polymorpha, there is still a lack of a highly efficient gene disruption method. To help address this issue, we presented a simple and efficient method for both single and multiple gene disruptions in H. polymorpha. The knockout system combined a variation of sticky-end polymerase chain reaction method (SEP), split marker deletion method, co-transformation of single-stranded DNA and mutant Cre-loxP system. Using a slightly modified LiAc/SS-DNA/PEG procedure, the co-transformation double-stranded split marker constructs together with single-stranded split marker constructs resulted in at least 70% homologous recombination events when the homologous genomic DNA fragment had a size of approximately 500bp. Our evidence suggested that single-stranded DNA may be responsible for the increased gene disruption efficiency. We demonstrated the effectiveness of the method for gene disruption by constructing both single and double gene disruptions at the ALG3 and URA5 loci in the same genetic background. The method described here presents an improved strategy for gene disruption and a potential application for investigation of biological processes in other yeast strains.

Expression of bovine follicle-stimulating hormone subunits in a Hansenula polymorpha expression system increases the secretion and bioactivity in vivo.

Bovine follicle-stimulating hormone (bFSH), a pituitary gonadotropin, is a heterodimer hormone that consists of a common alpha-subunit non-covalently associated with the hormone-specific beta-subunit. Unfortunately, expression levels of recombinant bFSH or its subunits are invariably low. We report here the secretory expression of biologically active bFSHalpha and bFSHbeta subunit in the methylotrophic yeast Hansenula polymorpha. A slightly higher level of expression of recombinant bFSH subunits was achieved by using the Saccharomyces cerevisiae-derived calnexin (ScCne1) as a chaperone in engineered H. polymorpha strains. The preliminary data also suggested that bFSH subunits expressed in H. polymorpha appeared to be less-glycosylated. This isoform had been shown to be 80% increase in in vivo bioactivity compared with the hyperglycosylated Pichia pastoris-derived recombinant bFSHalpha/beta. More sophisticated applications of bFSH would profit from the assembled less-glycosylated heterodimer.