| Products/Services Used | Details | Operation |
|---|---|---|
| PCR Cloning and Subcloning | ... 2 Materials. 2.1 Cloning SteT into the pTET-GFP 11 Vector and Generation of SteT Random Mutants. 1. cDNA encoding SteT (Genscript) with optimized codons for E. coli transcription and translation . 2. Primers to amplify the SteT coding region for error-prone PCR . 3. ... | Get A Quote |
Obtaining enough quantity of recombinant membrane transport proteins with optimal purity and stability for structural studies is a remarkable challenge. In this chapter, we describe a protocol to engineer SteT, the amino acid transporter of Bacillus subtilis, in order to improve its heterologous expression in Escherichia coli and its stability in detergent micelles. We built a library of 70 SteT mutants, combining a random mutagenesis protocol with a split GFP assay as reporter of protein folding and membrane insertion. Mutagenesis was restricted to residues situated in the transmembrane domains. Improved versions of SteT were successfully identified after analyzing the expression yield and monodispersity in de... More
