Production of G protein-coupled receptors in an insect-based cell-free system.
Andrei SonnabendViola SpahnMarlitt StechAnne ZemellaChristoph SteinStefan KubickPublished in: Biotechnology and bioengineering (2017)
The biochemical analysis of human cell membrane proteins remains a challenging task due to the difficulties in producing sufficient quantities of functional protein. G protein-coupled receptors (GPCRs) represent a main class of membrane proteins and drug targets, which are responsible for a huge number of signaling processes regulating various physiological functions in living cells. To circumvent the current bottlenecks in GPCR studies, we propose the synthesis of GPCRs in eukaryotic cell-free systems based on extracts generated from insect (Sf21) cells. Insect cell lysates harbor the fully active translational and translocational machinery allowing posttranslational modifications, such as glycosylation and phosphorylation of de novo synthesized proteins. Here, we demonstrate the production of several GPCRs in a eukaryotic cell-free system, performed within a short time and in a cost-effective manner. We were able to synthesize a variety of GPCRs ranging from 40 to 133 kDa in an insect-based cell-free system. Moreover, we have chosen the μ opioid receptor (MOR) as a model protein to analyze the ligand binding affinities of cell-free synthesized MOR in comparison to MOR expressed in a human cell line by "one-point" radioligand binding experiments. Biotechnol. Bioeng. 2017;114: 2328-2338. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.
Keyphrases
- cell free
- living cells
- endothelial cells
- circulating tumor
- aedes aegypti
- fluorescent probe
- binding protein
- induced pluripotent stem cells
- induced apoptosis
- pluripotent stem cells
- single cell
- stem cells
- chronic pain
- protein protein
- randomized controlled trial
- oxidative stress
- emergency department
- signaling pathway
- heat shock protein
- cell proliferation
- small molecule
- meta analyses