Expression of glycoproteins bearing complex human-like glycans with galactose terminal in Hansenula polymorpha
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ORIGINAL PAPER
Expression of glycoproteins bearing complex human-like glycans with galactose terminal in Hansenula polymorpha Hui Wang • Hao-lei Song • Qian Wang Bing-sheng Qiu
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Received: 25 July 2012 / Accepted: 15 October 2012 / Published online: 8 November 2012 Ó Springer Science+Business Media Dordrecht 2012
Abstract 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 (Man3GlcNAc2) intermediate can be generated in endoplasmic reticulum in HpALG3 and HpALG11 doublemutant H. polymorpha. Here, we describe the further modification of the glycosylation pathway in this doubledefect 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 b-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 b-1,2-Nacetylglucosaminyltransferase II (rGnTII) and human b-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 Gal2GlcNAc2Man3GlcNAc2 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. H. Wang H. Song Q. Wang B. Qiu (&) Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, China e-mail: [email protected]
Keywords Hansenula polymorpha Glycosylation Glycosyltransferase Galactose Human-like glycans
Introduction Protein-based drugs, such as antibodies and cytokines, have become the largest class of newly approved drugs, and the majority of these protein-based drugs are glycoproteins (Sethuraman and Stadheim 2006). The yeast-based expression system has been a promising alternative to express these glycoproteins for its short fermentation time, high production yield and cheap operating cost (Werten et al. 1999). However, glycoproteins derived from yeast and fungal cannot be used for therapeutic purposes due to their high-mannose type N-glycan contents which cause immune reactions in human body (Ballou 1990). Hence, therapeutic glycoproteins should be expressed in heterologous hosts that produce low mannose type or human-like glycans. Post-translational modifications, such as asparaginelinked (N-linked) glycosylation, are essential for the proper folding, stability and bioactivit
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