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Screening for the expression of soluble recombinant protein in E. coli

  • Author(s): Doyle, Sharon A.
  • et al.
Abstract

Protein expression and purification have traditionally been time-consuming, case-specific endeavors, and are considered to be the greatest bottlenecks in most proteomics pipelines. Eschericia coli (E. coli) is the most convenient and cost-effective host, although optimal conditions for the expression of different proteins vary widely. Proteins vary in their structural stability, solubility and toxicity in this environment, resulting in differing rates of protein degradation, formation into insoluble inclusion bodies and cell death, thus affecting the amount of soluble protein that can be obtained from E. coli grown in culture. A variety of E. coli strains designed to address many of these problems have been made, by allowing for disulfide bond formation in the E. coli cytoplasm, more efficient usage of codons that are rarely found in E. coli genes, and a reduction of specific E. coli proteases. The T7 expression system has also helped to alleviate many problems associated with recombinant expression by allowing for tightly regulated induction of expression. However variability in the recombinant expression of proteins still exists. Induction strength and time, and growth temperature are just a few parameters that can have drastic effects on the amount of soluble protein that can be obtained from the E. coli host. Adding further complexity to this problem, affinity tags that are used as a means to standardize protein purification have varying effects on protein stability and solubility. The hexahistidine tag, which allows for protein to be adsorbed to Ni2+-charged resin, is often used because it is small and often does not need to be removed by protease digestion prior to downstream experimentation. Another useful tag is the maltose binding protein (MBP) tag, which allows for the protein to be captured with amylose resin, and can stabilize and solubilize the fusion partner. A variety of affinity tags, in addition to their placement at the N- or C- terminal end of the coding sequence, needs to be tested for optimal protein expression. To take full advantage of the variety of strategies developed to improve the expression of soluble protein in E. coli, a means to test easily and rapidly many growth parameters is necessary. This chapter describes a dot-blot expression screen to test the effects of growth and induction parameters on the yield of soluble protein. The expression screen is used to detect hexahistidine-tagged proteins expressed in E. coli, however it is adaptable for the detection of other affinity tags or fusion partners that have suitable antibodies available. In this example, induction time and temperature are tested, however, it can be used to test additional parameters such as affinity tag type and placement, E. coli host type, and growth medium formulations. Results of the screen may be used to segregate samples into groups for further parallel processing in an efficient high-throughput protein production pipeline.

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