Genetic selection of solubility-enhanced proteins using the twin-arginine translocation system.
The expression of heterologous proteins in robust hosts such as Escherichia coli is often plagued by the tendency of the protein of interest to misfold and aggregate. To engineer and improve the folding properties of virtually any protein of interest, the quality control process inherent to the bacterial twin- arginine translocation (Tat) pathway can be exploited. The Tat pathway preferentially transports folded substrates across the inner membrane of E. coli with remarkable quality control that can provide selection pressure for protein folding and solubility. By fusing desired proteins to the N-terminus of mature TEM-1 β-lactamase and using an N-terminal signal peptide to target the fusion to the Tat pathway, it is possible to perform genetic selections for folded, soluble proteins. Here, we present a method for exploiting the folding quality control process associated with the Tat pathway for engineering folding-enhanced proteins.
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA. adam.fisher@glycobia.com
Adam C
AC
Fisher
Mark A
MA
Rocco
Matthew P
MP
DeLisa
D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012995:Solubility; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D001120:Arginine; D001618:beta-Lactamases