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, eIF2alpha and eIF2alpha-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 eIF2alpha, and thereby increased protein synthesis. By using this improved system, biologically active human choriogonadotropin (hCG), a glycoprotein hormone consisting of alpha and beta subunits was successfully synthesized. In conclusion, the hybridoma extract supplemented with GADD34/K3L should become a useful tool to produce recombinant glycoproteins.
authors.affiliation: RIKEN Genomic Sciences Center, Yokohama, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Mikami
authors.forename: Tominari
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Imataka
mesh_terms: D002474:Cell-Free System; D006063:Chorionic Gonadotropin / Q000096:biosynthesis*; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D006825:Hybridomas / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D006063:Chorionic Gonadotropin; D006023:Glycoproteins; D011994:Recombinant Proteins