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


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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    ABSTRACT: Escherichia coli is by far the most widely used bacterial host for the production of membrane proteins. Usually, different strains, culture conditions and production regimes are screened for to design the optimal production process. However, these E. coli-based screening approaches often do not result in satisfactory membrane protein production yields. Recently, it has been shown that (i) E. coli strains with strongly improved membrane protein production characteristics can be engineered or selected for, (ii) many membrane proteins can be efficiently produced in E. coli-based cell-free systems, (iii) bacteria other than E. coli can be used for the efficient production of membrane proteins, and, (iv) membrane protein variants that retain functionality but are produced at higher yields than the wild-type protein can be engineered or selected for. This article is part of a Special Issue entitled:Protein trafficking & Secretion.
    Biochimica et Biophysica Acta 11/2013; 1843(8). DOI:10.1016/j.bbamcr.2013.10.023 · 4.66 Impact Factor
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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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    ABSTRACT: DNA sequences are now far more readily available in silico than as physical DNA. De novo gene synthesis is an increasingly cost-effective method for building genetic constructs, and effectively removes the constraint of basing constructs on extant sequences. This allows scientists and engineers to experimentally test their hypotheses relating sequence to function. Molecular biologists, and now synthetic biologists, are characterizing and cataloging genetic elements with specific functions, aiming to combine them to perform complex functions. However, the most common purpose of synthetic genes is for the expression of an encoded protein. The huge number of different proteins makes it impossible to characterize and catalog each functional gene. Instead, it is necessary to abstract design principles from experimental data: data that can be generated by making predictions followed by synthesizing sequences to test those predictions. Because of the degeneracy of the genetic code, design of gene sequences to encode proteins is a high-dimensional problem, so there is no single simple formula to guarantee success. Nevertheless, there are several straightforward steps that can be taken to greatly increase the probability that a designed sequence will result in expression of the encoded protein. In this chapter, we discuss gene sequence parameters that are important for protein expression. We also describe algorithms for optimizing these parameters, and troubleshooting procedures that can be helpful when initial attempts fail. Finally, we show how many of these methods can be accomplished using the synthetic biology software tool Gene Designer.
    Methods in Enzymology, 489 edited by Christopher Voigt, 01/2011: chapter 3: pages 43-66; Elsevier Inc.., ISBN: 978-0123851208
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