title: Translation levels control multi-spanning membrane protein expression.
abstract: Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli). These results demonstrate that excessive translation initiation rates of membrane proteins cause a block in protein synthesis and ultimately prevent the high-level accumulation of these proteins. Moderate translation rates allow coupling of peptide synthesis and membrane targeting, resulting in a significant increase in protein expression and accumulation over time. The current study evaluates four membrane proteins, CD20 (4-transmembrane (TM) helixes), the G-protein coupled receptors (GPCRs, 7-TMs) RA1c and EG-VEGFR1, and Patched 1 (12-TMs), and demonstrates the critical role of translation initiation rates in the targeting, insertion and folding of integral membrane proteins in the E. coli membrane.
authors.affiliation: Department of Antibody Engineering, Genentech Inc., South San Francisco, California, United States of America.
authors.forename: Hok Seon
authors.initials: HS
authors.lastname: Kim
authors.forename: James A
authors.initials: JA
authors.lastname: Ernst
authors.forename: Cecilia
authors.initials: C
authors.lastname: Brown
authors.forename: Jenny
authors.initials: J
authors.lastname: Bostrom
authors.forename: Germaine
authors.initials: G
authors.lastname: Fuh
authors.forename: Chingwei V
authors.initials: CV
authors.lastname: Lee
authors.forename: Arthur
authors.initials: A
authors.lastname: Huang
authors.forename: Richard L
authors.initials: RL
authors.lastname: Vandlen
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Yansura
mesh_terms: D018951:Antigens, CD20 / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D010442:Peptide Chain Initiation, Translational; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D018951:Antigens, CD20; D008565:Membrane Proteins; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins