Expression Library
Media Recipes
E. coli strains for expression
Vector Library
Relevant CSBMP Publications
An enlightening and informative tome on E. coli culturing for protein expression is the article by Frank Studier ( Studier FW: Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 2005, 41:207-234 ). This article is a "must-read" for those interested in protein expression in E. coli.
Media Recipes
Growth and expression studies at the CSBMP have often started with using the standard LB broth, 2TY broth, or the "Terrific" broth. The recipes for our versions of these media can be found here.
However, a series of growth media recipes based on the standard LB broth, enhanced through the addition of glucose, metal salts, and/or glycerol in order to mimic the yeast extract-based recipes described by Studier (see above reference). Some of these recipes have been useful for achieving increase cell mass, increasing the level of expression, and reducing leakiness of some expression vectors. The recipes for our versions of these media can be found here.
As many strains of E. coli, including most BL21 (DE3) derivatives, grow better in fermentors using minimal media rather than the richer yeast-extract broths (R. M. Worden and J. Sun, personal observations), several specially-designed minimal media are recommended by our local facility for large-scale fermentation.
E. coli strains for expression
As our base vector had a T7 promoter site, we decided to use only E. coli BL21 (DE3) derivative strains, of which many are commercially available including the C41 (DE3) and C43 (DE3) strains developed in the Walker laboratory [1-3]. The IPTG-inducible protein expression with the vector however remains even if the T7 promoter is replaced by the weaker, quite efficient tac promoter.
1. Miroux B, Walker JE: Over-production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. Journal of Molecular Biology 1996, 260:289-298.
2. Arechaga I, Miroux B, Karrasch S, Huijbregts R, de Kruijff B, Runswick MJ, Walker JE: Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase. FEBS Lett 2000, 482:215-219.
3. Arechaga I, Miroux B, Runswick MJ, Walker JE: Over-expression of Escherichia coli F1F(o)-ATPase subunit a is inhibited by instability of the uncB gene transcript. FEBS Lett 2003, 547:97-100.
Vector Library
We have been designing and optimizing expression vectors to support high-yield expression of some membrane proteins. Although the number of possible choices is quite large, we focused primarily with pLW01, a small, mulitcopy expression vector developed by Lucy Waskell at the University of Michigan. The pLW01 expression vector [4] has a standard T7 promoter site and uses ampicillin resistance for selection. This vector has been quite effective in expressing single transit membrane proteins, like native mammalian cytochrome P450s [4-6] and cytochrome b5 [7], with yields as high as 50 mg of purified protein per liter of culture. Information about the pLW01 vector can be found here.
Using the pLW01 plasmid as a template, we made modifications to explore how “metabolic” burden [8], mRNA stability [9], translation efficiency [10,11], and membrane integration [12] impacted membrane protein expression. About 60 different vectors were made for expression studies based on the pLW01 parent. The some of these new vectors, designated pRMG, can be found here.
4. Bridges A, Gruenke L, Chang YT, Vakser IA, Loew G, Waskell L: Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase. J Biol Chem 1998, 273:17036-17049.
5. Cheng D, Kelley RW, Cawley GF, Backes WL: High-level expression of recombinant rabbit cytochrome P450 2E1 in Escherichia coli C41 and its purification. Protein Expr Purif 2004, 33:66-71.
6. Saribas AS, Gruenke L, Waskell L: Overexpression and purification of the membrane-bound cytochrome P450 2B4. Protein Expr Purif 2001, 21:303-309.
7. Mulrooney SB, Waskell L: High-level expression in Escherichia coli and purification of the membrane-bound form of cytochrome b(5). Protein Expr Purif 2000, 19:173-178.
8. Soriano E, Borth N, Katinger H, Mattanovich D: Flow Cytometric Analysis of Metabolic Stress Effects Due to Recombinant Plasmids and Proteins in Escherichia coli Production Strains. Metab Engineering 1999, 1:270-274.
9. Iost I, Dreyfus M: The stability of Escherichia coli lacZ mRNA depends upon the simultaneity of its synthesis and translation. Embo J 1995, 14:3252-3261.
10. Etchegaray JP, Inouye M: Translational enhancement by an element downstream of the initiation codon in Escherichia coli. J Biol Chem 1999, 274:10079-10085.
11. Golshani A, Krogan NJ , Xu J, Pacal M, Yang XC, Ivanov I, Providenti MA, Ganoza MC, Ivanov IG, AbouHaidar MG: Escherichia coli mRNAs with strong Shine/Dalgarno sequences also contain 5' end sequences complementary to domain # 17 on the 16S ribosomal RNA. Biochem Biophys Res Commun 2004, 316:978-983.
12. Hessa T, Kim H, Bihlmaier K, Lundin C, J B, Andersson H, Nilsson I, White SH, von Heijne G: Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 2005, 433:377-381.
Relevant CSBMP Publications
Distler AM, Hiser C, Qin L, Hilmi Y, Ferguson-Miller S, Allison J: Mass spectrometric detection of protein, lipid, and heme components of cytochrome c oxidase from R. sphaeroides and the stabilization of non-covalent complexes from the Enzyme. Eur. J. Mass Spectrom. 2004, 10:295-308.
Qin L, Hiser C, Mulichak AM, Garavito RM, Ferguson-Miller S: Identification of conserved lipid detergent-binding sites in a high-resolution structure of the membrane protein cytochrome coxidase. Proc Natl Acad Sci U S A 2006, 103:16117-16122.