Choline-binding domain as a novel affinity tag for purification of fusion proteins produced in Pichia pastoris

Biotechnol Bioeng. 2001 Jul 20;74(2):164-71. doi: 10.1002/bit.1106.

Abstract

The choline-binding domain (ChoBD) of the carboxy-terminal region of the Streptococcus pneumoniae amidase LYTA (C-LYTA) presents a strong affinity for tertiary amines. We report a method for single-step purification of proteins expressed in the methylotrophic yeast Pichia pastoris based on the fusion of C-LYTA to the protein of interest. We show that C-LYTA can be efficiently expressed and secreted in this host. Tagged proteins fused to this binding domain can be purified on inexpensive DEAE matrices. It therefore provides a useful system for the purification of recombinant proteins with high specificity suitable for industrial purposes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites
  • Choline / metabolism*
  • Chromatography, DEAE-Cellulose / methods*
  • Molecular Sequence Data
  • N-Acetylmuramoyl-L-alanine Amidase / genetics*
  • N-Acetylmuramoyl-L-alanine Amidase / isolation & purification
  • N-Acetylmuramoyl-L-alanine Amidase / metabolism
  • Pichia / genetics*
  • Pichia / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification*
  • Recombinant Fusion Proteins / metabolism*
  • Streptococcus pneumoniae / enzymology
  • Streptococcus pneumoniae / genetics
  • beta-Lactamases / genetics
  • beta-Lactamases / isolation & purification
  • beta-Lactamases / metabolism

Substances

  • Recombinant Fusion Proteins
  • EJL amidase
  • N-Acetylmuramoyl-L-alanine Amidase
  • beta-Lactamases
  • Choline