Record Details

Engineering the N-Glycosylation Pathway in Pichia Pastoris for the Expression of Glycoprotein Hormones

Electronic Theses of Indian Institute of Science

View Archive Info
 
 
Field Value
 
Title Engineering the N-Glycosylation Pathway in Pichia Pastoris for the Expression of Glycoprotein Hormones
 
Creator Manoharan, Simna
 
Subject Glycoprotein Hormones
N-Glycosylation
Recombinant Protein Expression
Pichia Pastoris N-Glycosylation
Industrial Proteins
Glycosylated Protein Expression
Protein Glycosylation
Glycoengineered Pichia Pastoris
Human Chorionic Gonadotropin (hCG)
Follicle Stimulating Hormone (FSH)
Therapeutic Proteins
Glycoprotein Hormone Expression
Recombinant Therapeutic Proteins
Pichia pastoris
Glycoengineered Strains
Chemical Engineering
 
Description Proteins, participating in a myriad of biological function, are at the core of all cellular activities occurring within living organisms. Therapeutic proteins, hence constitute a major part of the pharmaceutical industry. The glycoprotein hormones follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid stimulating hormone (TSH) and human chorionic gonadotropin (CG) regulate various reproductive and metabolic functions in humans and hence have high therapeutic potentials. The increasing demand of recombinant proteins for therapeutic uses drives the development of better expression systems.
The methylotrophic yeast Pichia pastoris, has been termed as an industrial workhorse for heterologous protein expression. However, the N-glycosylation in yeast is of the high mannose type, resulting in a reduced serum half-life of the recombinant proteins. In the current work, we have re-engineered the Pichia N-glycosylation pathway to mimic the human type of N-glycosylation. Towards this end, we abolished the yeast native N-glycosylation and introduced enzymes from various eukaryotic sources into the system. These modifications resulted in the conversion of the yeast Man9-20GlcNAc2 glycan structure to a more human like GlcNAc2Man3GlcNAc2 form on over 70 % of the heterologous expressed proteins.
In order to demonstrate the application of these strains as efficient protein expression hosts, the glycoengineerd Pichia was used for large scale expression of the glycoprotein hormones, hCG and FSH. The purified recombinant hormones were found to have binding affinities and structure similar to that of the natural hormones. These recombinant hormones were also able to elicit over two fold responses in animal models compared to buffer controls and the activity was comparable to the natural counterparts. Thus, we report the generation of a glycoengineered Pichia pastoris, which can be considered as a serious contender for the expression of glycosylated proteins of therapeutic importance.
 
Contributor Modak, Jayant M
 
Date 2018-01-15T05:05:15Z
2018-01-15T05:05:15Z
2018-01-15
2016
 
Type Thesis
 
Identifier http://hdl.handle.net/2005/3017
http://etd.ncsi.iisc.ernet.in/abstracts/3880/G28323-Abs.pdf
 
Language en_US
 
Relation G28323