An evolved Mxe GyrA intein for enhanced production of fusion proteins

ACS Chem Biol. 2015 Feb 20;10(2):527-38. doi: 10.1021/cb500689g. Epub 2014 Nov 24.

Abstract

Expressing antibodies as fusions to the non-self-cleaving Mxe GyrA intein enables site-specific, carboxy-terminal chemical modification of the antibodies by expressed protein ligation (EPL). Bacterial antibody-intein fusion protein expression platforms typically yield insoluble inclusion bodies that require refolding to obtain active antibody-intein fusion proteins. Previously, we demonstrated that it was possible to employ yeast surface display to express properly folded single-chain antibody (scFv)-intein fusions, therefore permitting the direct small-scale chemical functionalization of scFvs. Here, directed evolution of the Mxe GyrA intein was performed to improve both the display and secretion levels of scFv-intein fusion proteins from yeast. The engineered intein was shown to increase the yeast display levels of eight different scFvs by up to 3-fold. Additionally, scFv- and green fluorescent protein (GFP)-intein fusion proteins can be secreted from yeast, and while fusion of the scFvs to the wild-type intein resulted in low expression levels, the engineered intein increased scFv-intein production levels by up to 30-fold. The secreted scFv- and GFP-intein fusion proteins retained their respective binding and fluorescent activities, and upon intein release, EPL resulted in carboxy-terminal azide functionalization of the target proteins. The azide-functionalized scFvs and GFP were subsequently employed in a copper-free, strain-promoted click reaction to site-specifically immobilize the proteins on surfaces, and it was demonstrated that the functionalized, immobilized scFvs retained their antigen binding specificity. Taken together, the evolved yeast intein platform provides a robust alternative to bacterial intein expression systems.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Click Chemistry
  • DNA Gyrase / chemistry*
  • DNA Gyrase / metabolism
  • Directed Molecular Evolution*
  • Inteins / physiology*
  • Models, Molecular
  • Molecular Structure
  • Protein Conformation
  • Protein Folding
  • Recombinant Proteins*
  • Saccharomyces cerevisiae / metabolism
  • Small Molecule Libraries

Substances

  • Recombinant Proteins
  • Small Molecule Libraries
  • DNA Gyrase