Protein Expression in E.coli Cell-free System

Alpha Lifetech Inc. focuses on recombinant proteins' expression in different Biosystems for many years, including prokaryotic and eukaryotic organisms, and also for the development of novel high-potency recombinant proteins.


Cell-free Protein Expression System---Membrane Protein (MPs) Expression in E. coli Extracts


Alpha Lifetech Inc. provides clients with a reliable expression of membrane proteins to meet native configuration and functional requirements by using a cell-free protein expression system. Conventional methods, such as Mammal Expression System, Insect Expression System, Bacteria Expression System and Yeast Expression System, for the preparation of integral membrane proteins have intrinsic limits in membrane proteins' expression, because the production of membrane proteins is a challenging task as their hydrophobic nature and their specific requirements in cellular expression systems frequently prevent an efficient synthesis. So far, a considerable number of membrane proteins from diverse families like prokaryotic small multidrug transporters (SMTs) or eukaryotic G-protein coupled receptors (GPCRs) have been produced in cell-free systems in high amounts and in functionally active forms. Compared to the Wheat Germ Cell-free Expression System and other Mammal Cell-free Expression System, the E. coli Cell-free Expression System is well suited for the expression of prokaryotic MPs. 


Principle of E. coli Cell-free Expression System


Experts of Alpha Lifetech Inc. have modified and constructed a powerful vector system that contained double plasmids: one regulates the expression of T7 RNA polymerase inhibitor protein; another one encodes transfer RNAs for rare E. coli codons. Alpha Lifetech Inc. deploys an ingredients-defined E. coli Cell-free Expression System for expressing each client's target protein.

Fig. 1 Mode A or B Represent Insoluble or Soluble Form of Membrane Proteins separately in Cell-free systems


MPs can be expressed as a precipitate (Mode A), solubilized in an appropriate detergent and further reconstituted into proteoliposomes, or they can be soluble expressed in the presence of detergent (Mode B), purified and reconstituted into proteoliposomes. AlphaBacTM, bacterial in vitro expression systems use an efficient coupled transcription and translation reaction to produce high yields of full-length, functional protein from purified E. coli cell lysates.

Service Features:

-- High Productivity: mg magnitude

-- High Solution for Membrane Protein

-- Toxic Protein Produce

-- Free Modification

-- CD-Spectroscopy

-- Solution-NMR Spectroscopy

-- Crystallography

-- Ligand interaction analysis

-- Single particle analysis

-- Pull-down experiments/ Binding assays

-- For oligomerisation studies

For each project, we will provide a detailed design based on the characteristics of different membrane proteins. Besides, Alpha Lifetech Inc. develops other cell-free systems for eukaryotic MPs expression.