Expression and secretion of glycosylated heparin biosynthetic enzymes using Komagataella pastoris
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Heparin, an anticoagulant drug, is biosynthesized in selected animal cells. The heparin biosynthetic enzymes mainly consist of sulfotransferases and all are integral transmembrane glycoproteins. These enzymes are generally produced in engineered Escherichia coli as without their transmembrane domains as non-glycosylated fusion proteins. In this study, we used the yeast, Komagataella pastoris, to prepare four sulfotransferases involved in heparin biosynthesis as glycoproteins. While the yields of these yeast-expressed enzymes were considerably lower than E. coli-expressed enzymes, these enzymes were secreted into the fermentation media simplifying their purification and were endotoxin free. The activities of these sulfotransferases, expressed as glycoproteins in yeast, were compared to the bacterially expressed proteins. The yeast-expressed sulfotransferase glycoproteins showed improved kinetic properties than the bacterially expressed proteins.
KeywordsHeparin Biosynthetic enzymes Yeast expression Bacterial expression Kinetics
The authors gratefully acknowledge funding from the National Institutes of Health (HL096972), the National Science Foundation (MCB-1448657), and funding from the China Scholarship Council.
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare that they have no conflict of interest.
- Biedendieck R, Beine R, Gamer M, Jordan E, Buchholz K, Seibel J, Dijkhuizen L, Malten M, Jahn D (2007) Export, purification, and activities of affinity tagged Lactobacillus reuteri levansucrase produced by Bacillus megaterium. Appl Microbiol Biotechnol 74:1062–1073. doi: 10.1007/s00253-006-0756-0 CrossRefPubMedGoogle Scholar
- de Oliveira Magalhães P, Lopes AM, Mazzola PG, Rangel-Yagui C, Penna TCV, Pessoa A (2007) Methods of endotoxin removal from biological preparations: a review. J Pharm Pharm Sci 10:388–404Google Scholar
- Kazenwadel C, Klebensberger J, Richter S, Pfannstiel J, Gerken U, Pickel B, Schaller A, Hauer B (2013) Optimized expression of the dirigent protein AtDIR6 in Pichia pastoris and impact of glycosylation on protein structure and function. Appl Microbiol Biotechnol 97:7215–7227. doi: 10.1007/s00253-012-4579-x CrossRefPubMedGoogle Scholar
- Opal SM, Scannon PJ, Vincent JL, White M, Carroll SF, Palardy JE, Parejo NA, Pribble JP, Lemke JH (1999) Relationship between plasma levels of lipopolysaccharide (LPS) and LPS-binding protein in patients with severe sepsis and septic shock. J Infect Dis 180:1584–1589. doi: 10.1086/315093 CrossRefPubMedGoogle Scholar
- Pla IA, Damasceno LM, Vannelli T, Ritter G, Batt CA, Shuler ML (2006) Evaluation of Mut+ and MutS Pichia pastoris phenotypes for high level extracellular scFv expression under feedback control of the methanol concentration. Biotechnol Prog 22:881–888. doi: 10.1021/bp060012+ CrossRefPubMedGoogle Scholar
- Pyati P, Fitches E, Gatehouse JA (2014) Optimising expression of the recombinant fusion protein biopesticide ω-hexatoxin-Hv1a/GNA in Pichia pastoris: sequence modifications and a simple method for the generation of multi-copy strains. J Ind Microbiol Biotechnol 41:1237–1247. doi: 10.1007/s10295-014-1466-8 CrossRefPubMedGoogle Scholar
- Shirke AN, Basore D, Holton S, Su A, Baugh E, Butterfoss GL, Makhatadze G, Bystroff C, Gross RA (2016b) Influence of surface charge, binding site residues and glycosylation on Thielavia terrestris cutinase biochemical characteristics. Appl Microbiol Biotechnol 100:4435–4446. doi: 10.1007/s00253-015-7254-1 CrossRefPubMedGoogle Scholar