Co-expression of molecular chaperones does not improve the heterologous expression of mammalian G-protein coupled receptor expression in yeast
| Autor: | James A. Butz, Ronald T. Niebauer, Anne S. Robinson |
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| Rok vydání: | 2003 |
| Předmět: |
Receptor
Adenosine A2A G protein Bioengineering Saccharomyces cerevisiae Protein Engineering Applied Microbiology and Biotechnology Substance-P Receptor Receptors G-Protein-Coupled Mice Gene Expression Regulation Fungal Animals Humans Protein disulfide-isomerase Receptor Integral membrane protein G protein-coupled receptor Mammals biology Receptors Neurokinin-1 Recombinant Proteins Rats Biochemistry Chaperone (protein) biology.protein Heterologous expression Biotechnology Molecular Chaperones |
| Zdroj: | Biotechnology and bioengineering. 84(3) |
| ISSN: | 0006-3592 |
| Popis: | The limitations to high-level expression of integral membrane proteins are not well understood. The human A(2)a adenosine receptor (A(2)a) and mouse Substance P receptor (SPR) were individually expressed in S. cerevisiae to identify potential cellular bottlenecks for G-protein coupled receptors. In the yeast system, A(2)a was not N-linked glycosylated but was functional and plasma membrane-localized. A(2)a also contained an intramolecular disulfide bond. Substance P receptor was also not N-linked glycosylated in yeast, but, unlike A(2)a, SPR was intracellularly retained, nonfunctional, and did not appear to contain an intramolecular disulfide bond. Since both receptors contain N-linked glycosylation and disulfide bonds in mammalian systems, machinery responsible for interacting with these modifications was investigated-specifically, the potential interactions between the nascent receptor and ER-resident proteins were explored. The chaperones calnexin and protein disulfide isomerase were co-overexpressed with the GPCRs to determine the effect on total and active yields of A(2)a and SPR, as well as on receptor trafficking. The effect of co-expressing the chaperone BiP on the total yields of A(2)a as well as intracellular fates of both receptors were determined. The co-expression of ER resident proteins did not improve A(2)a yields nor did they restore SPR activity or improve SPR cell surface expression. Taken together, these results indicate that an ER-folding bottleneck does not limit the expression of the mammalian receptors in yeast. |
| Databáze: | OpenAIRE |
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