Article

Rational design of a fusion partner for membrane protein expression in E

New England Biolabs, Inc. Gene Expression Division, Ipswich, Massachusetts 01938, USA.
Protein Science (Impact Factor: 2.85). 08/2009; 18(8):1735-44. DOI: 10.1002/pro.189
Source: PubMed

ABSTRACT

We have designed a novel protein fusion partner (P8CBD) to utilize the co-translational SRP pathway in order to target heterologous proteins to the E. coli inner membrane. SRP-dependence was demonstrated by analyzing the membrane translocation of P8CBD-PhoA fusion proteins in wt and SRP-ffh77 mutant cells. We also demonstrate that the P8CBD N-terminal fusion partner promotes over-expression of a Thermotoga maritima polytopic membrane protein by replacement of the native signal anchor sequence. Furthermore, the yeast mitochondrial inner membrane protein Oxa1p was expressed as a P8CBD fusion and shown to function within the E. coli inner membrane. In this example, the mitochondrial targeting peptide was replaced by P8CBD. Several practical features were incorporated into the P8CBD expression system to aid in protein detection, purification, and optional in vitro processing by enterokinase. The basis of membrane protein over-expression toxicity is discussed and solutions to this problem are presented. We anticipate that this optimized expression system will aid in the isolation and study of various recombinant forms of membrane-associated protein.

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Available from: James C Samuelson, May 05, 2014
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    • "This results in improved cell growth and a twofold increase in membrane protein production yields. Tuning translation rates by using expression vectors with ribosome binding sites of different strength can also be used to improve membrane protein production yields [61]. Finally, expression vectors encoding small N-terminal fusion tags with different translation initiation rates have also been used to improve membrane protein production yields [62] "
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    • "Transmembrane proteins can be particularly difficult to successfully express in heterologous hosts (Freigassner et al., 2009). Quite often such proteins are poorly directed to the membrane and often are toxic to the cell (Luo et al., 2009; Steffensen and Pedersen, 2006; Wagner et al., 2006, 2008). For both reasons, attenuated expression constructs may prove useful (Wagner et al., 2008). "
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