29.01.2009

Fourteen laboratories, 83 authors, and over 10,000 recombinant proteins:

A consensus on effective methods for protein expression and purification in E. coli

When it comes to recombinant protein expression, it can be tremendously helpful to listen to collective wisdom. In this review published in Nature Methods*, there is no shortage of expert advice. Eighty-three authors from 14 high-throughput laboratories - all focused on protein production for structural genomics - have collaborated to author an overview of recombinant protein expression in E. coli. In the process of discussing cloning strategies, expression host strains, promoters, affinity tags, and purification methods, the authors have articulated a “Consensus Protocol” of methods that work well for the majority of recombinant proteins.

Although the laboratories differ in their approaches, several patterns emerge. With regard to cloning methodologies, nine out of 14 groups rely partially or totally on Ligation-Independent Cloning (LIC). Twelve out of 14 facilities utilize vectors in which expression is controlled by T7 RNA polymerase, as is the case with the pET vector series. Consequently, the most frequently recommended expression host strain is BL21(DE3), a λDE3 lysogen of BL21 that supports T7-mediated expression and is deficient in lon and ompT proteases for improved target protein stability. Many of the laboratories dedicated to expression of eukaryotic genes in E. coli have successfully employed expression host strains carrying plasmids encoding tRNAs rarely used in E. coli, such as Rosetta^™ (DE3) and Rosetta 2 (DE3) strains. Cell lysis and sample handling is often improved using reagents such as Benzonase^® nuclease and BugBuster^® Protein Extraction Reagent. For cell culture, auto-inducing media has improved the efficiency of many high-throughput expression projects. Media of this type – such as Overnight Express^™ Autoinduction System– gradually elicit protein expression through metabolic shift without the addition of an artificial inducing agent such as IPTG. And with regard to affinity tags and purification strategies, the overwhelming choice was the use of hexahistidine tags (such as His•Tag^® fusions) followed by immobilized metal affinity chromatography (IMAC) purification. The collective experience of the authors is tremendous. In the past ten years, they have cloned a total of 109,443 targets and purified 28,393 recombinant proteins. Thus, the review focuses on practical logistical advice presented in a way that is highly accessible to researchers with little experience in protein expression. Novagen^® products have been at the forefront of the protein expression field for nearly 20 years, and we are honoured to contribute to the success of projects of every size and scope. Please refer to the table below for a summary of key Novagen protein expression products.

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