Engineered calcium-precipitable restriction enzyme

ACS Synth Biol. 2014 Dec 19;3(12):969-71. doi: 10.1021/sb500042m.

Abstract

We have developed a simple system for tagging and purifying proteins. Recent experiments have demonstrated that RTX (Repeat in Toxin) motifs from the adenylate cyclase toxin gene (CyaA) of B. pertussis undergo a conformational change upon binding calcium, resulting in precipitation of fused proteins and making this method a viable alternative for bioseparation. We have designed an iGEM Biobrick comprised of an RTX tag that can be easily fused to any protein of interest. In this paper, we detail the process of creating an RTX tagged version of the restriction enzyme EcoRI and describe a method for expression and purification of the functional enzyme.

Publication types

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

MeSH terms

  • Adenylate Cyclase Toxin / genetics
  • Amino Acid Motifs / genetics*
  • Calcium / chemistry
  • Calcium / metabolism*
  • Cloning, Molecular
  • Deoxyribonuclease EcoRI / genetics*
  • Genetic Engineering / methods*
  • Protein Conformation
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification*
  • Recombinant Fusion Proteins / metabolism*

Substances

  • Adenylate Cyclase Toxin
  • Recombinant Fusion Proteins
  • Deoxyribonuclease EcoRI
  • Calcium