Fusion order controls expression level and activity of elastin-like polypeptide fusion proteins

Trine Christensen, Miriam Amiram, Sue Dagher, Kimberly Trabbic-Carlson, Mohammed F. Shamji, Lori A. Setton, Ashutosh Chilkoti

Research output: Contribution to journalArticlepeer-review

63 Scopus citations

Abstract

We have previously developed a method to purify recombinant proteins, termed inverse transition cycling (ITC) that eliminates the need for column chromatography. ITC exploits the inverse solubility phase transition of an elastin-like polypeptide (ELP) that is fused to a protein of interest. In ITC, a recombinant ELP fusion protein is cycled through its phase transition, resulting in separation of the ELP fusion protein from other Escherichia coli contaminants. Herein, we examine the role of the position of the ELP in the fusion protein on the expression levels and yields of purified protein for four recombinant ELP fusion proteins. Placing the ELP at the C-terminus of the target protein (protein-ELP) results in a higher expression level for the four ELP fusion proteins, which also translates to a greater yield of purified protein. The position of the fusion protein also has a significant impact on its specific activity, as ELP-protein constructs have a lower specific activity than protein-ELP constructs for three out of the four proteins. Our results show no difference in mRNA levels between protein-ELP and ELPprotein fusion constructs. Instead, we suggest two possible explanations for these results: first, the translational efficiency of mRNA may differ between the fusion protein in the two orientations and second, the lower level of protein expression and lower specific activity is consistent with a scenario that placement of the ELP at the N-terminus of the fusion protein increases the fraction of misfolded, and less active conformers, which are also preferentially degraded compared to fusion proteins in which the ELP is present at the C-terminal end of the protein.

Original languageEnglish
Pages (from-to)1377-1387
Number of pages11
JournalProtein Science
Volume18
Issue number7
DOIs
StatePublished - 1 Jul 2009
Externally publishedYes

Keywords

  • Elastin-like polypeptides
  • Fusion order
  • Fusion proteins
  • Inverse transition cycling
  • Protein yields
  • Specific activities

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