NYCOMPS

Protein Production

NYCOMPS uses high-throughput automated methods to clone, express and assess the suitability for structural studies of hundreds of NYCOMPS has established a high throughput pipeline specifically for integral membrane proteins based on much of the groundbreaking work designed for structural genomics of soluble proteins. The NYCOMPS pipeline is the first structural genomics pipeline specifically for membrane proteins that has processed thousands of targets. Targets identified by the bioinformatic section are amplified by PCR on the 384 well scale using a bank of 96 bacterial genomic DNA’s as template material. Amplified DNA is purified robotically and inserted into bacterial expression vectors, designed by NYCOMPS, via ligation independent cloning. Bacteria are transformed and are robotically plated onto 24 well blocks (each containing 2mls of selective agar media). Transformants are picked, and grown in specialized high growth density incubators and expression plasmids are isolated using our liquid handling robot (approximately 384 ‘minipreps’ every 2 hours). Expression vectors are verified by sequencing and samples are sent to the PSI Material Repository. Correctly sequenced expression vectors are transfered to expression strains (typically BL21 (DE3) pLysS -T1R) and grown on a 96 well scale for expression testing. Pellets are harvested and lysed by sonication using the NYCOMPS sonicator robot. Whole cell lysates are solubilized using 2% DDM detergent, and membrane proteins are extracted via their deca histidine tag that is fused to the N or C terminus, this tag having been engineered in place from the expression vector backbone. Metal affinity chromatography is conducted (in 0.02% DDM) and eluted proteins are assayed by SDS-PAGE and coomassie staining.

Proteins that express well are scaled up to a midi scale via growth in the GNF fermenter and are purified using a similar scheme to the small scale preparations. Eluted proteins from the medium scale expression are assayed for detergent preference via HPLC size exclusion chromatography. This process involves the injection of a small (5 micro liter) purified sample of the membrane protein onto a microbore silica column and monitoring the elution profile. The mobile phase of the column is supplemented with detergent at ~twice the CMC of the detergent. After all the protein injections have been made in the first mobile phase/detergent combination, the column is equilibrated into the second detergent solution and the injections repeated. This is then carried out for all further detergents, and an informative array of elution peak profiles versus detergent type is built up. This informs the investigators as to which detergents should be used in large scale purifications and crystallization trials.

Watch the webinar: Strategies for producing membrane proteins in E. coli