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A hybridoma-based in vitro translation system that efficiently synthesizes glycoproteins

Paper ID Volume ID Publish Year Pages File Format Full-Text
25560 43581 2006 14 PDF Available
Title
A hybridoma-based in vitro translation system that efficiently synthesizes glycoproteins
Abstract

Since a large number of eukaryotic proteins are glycoproteins, an efficient and easily available cell-free system for the production of recombinant glycoproteins is needed. We have successfully developed an efficient cell-free translation system derived from a monoclonal antibody-producing hybridoma for this purpose. While extracts from HeLa cells were very inefficient for production of an N-glycosylated form of human immunodeficiency virus type-1 envelope protein 120 (gp120), the hybridoma extract was able to fully N-glycosylate gp120. During cell-free translation, eIF2α and eIF2α-kinases in the hybridoma extracts were observed to become phosphorylated due to the presence of essential supplements creatine phosphate and ATP. Addition of recombinant GADD34 and/or K3L to the extract efficiently lowered the phosphorylation of eIF2α, and thereby increased protein synthesis. By using this improved system, biologically active human choriogonadotropin (hCG), a glycoprotein hormone consisting of α and β subunits was successfully synthesized. In conclusion, the hybridoma extract supplemented with GADD34/K3L should become a useful tool to produce recombinant glycoproteins.

Keywords
Cell-free protein synthesis; Glycoprotein; Translation; Hybridoma; N-Glycosylation
First Page Preview
A hybridoma-based in vitro translation system that efficiently synthesizes glycoproteins
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 127, Issue 1, 15 December 2006, Pages 65–78
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering