A PCR-directed cell-free approach to optimize protein expression using diverse fusion tags

Andrew V Kralicek, Mazdak Radjainia, Nazratul A.B. Mohamad Ali, Colm Carraher, Richard D Newcomb, Alok K Mitra

Research output: Contribution to journalArticleResearchpeer-review

12 Citations (Scopus)

Abstract

N-terminal fusion tags that enhance translation initiation or protein solubility are often used to facilitate protein overexpression. As the optimal tag for a given target protein cannot be predicted a priori, valuable time can be lost in cloning and manipulating the corresponding gene to generate different fusion constructs for expression analysis. We have developed a cell-free strategy that consolidates these steps, enabling the utility of a panel of nine fusion-tags to be determined within one to two days. This approach exploits the fact that PCR-amplified DNA can be used as a template for cell-free protein synthesis. Overlap/extension PCR using the TEV protease site as the overlap region allows the fusion of different T7 promoter (T7p)-tag-TEV DNA fragments with a TEV-gene-T7 terminator (T7ter) fragment. For tag sequences where the TEV site is not compatible, a short C 3G 3 repeat (CGr) sequence can be used as the overlap region. The resulting T7p-tag-TEV-gene-T7ter constructs are then used as templates for PCR-directed cell-free protein synthesis to identify which tag-TEV-gene fusion protein produces the highest amount of soluble protein. We have successfully applied this approach to the overexpression of the Adiponectin hypervariable domain (AHD). Five of the nine N-terminal fusion tags tested enabled the synthesis of soluble recombinant protein. The best of these was the Peptidyl-prolylcis-trans isomerise B (PpiB) fusion tag which produces 1 mg/ml amounts of soluble fusion protein. PpiB is an example of a new class of fusion tag known as the "stress-responsive proteins". Our results suggest that this cell-free fusion-tag expression screen facilitates the rapid identification of suitable fusion-tags that overcome issues such as poor expression and insolubility, often encountered using conventional approaches.

Original languageEnglish
Pages (from-to)117-124
Number of pages8
JournalProtein Expression and Purification
Volume80
Issue number1
DOIs
Publication statusPublished - Nov 2011
Externally publishedYes

Keywords

  • Adiponectin
  • Cell-free protein synthesis
  • Fusion tag
  • Overlap/extension PCR
  • Peptidyl-prolyl-isomerase B

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