The pCri System: a vector collection for recombinant protein expression and purification

PLoS One. 2014 Nov 11;9(11):e112643. doi: 10.1371/journal.pone.0112643. eCollection 2014.

Abstract

A major bottleneck in structural, biochemical and biophysical studies of proteins is the need for large amounts of pure homogenous material, which is generally obtained by recombinant overexpression. Here we introduce a vector collection, the pCri System, for cytoplasmic and periplasmic/extracellular expression of heterologous proteins that allows the simultaneous assessment of prokaryotic and eukaryotic host cells (Escherichia coli, Bacillus subtilis, and Pichia pastoris). By using a single polymerase chain reaction product, genes of interest can be directionally cloned in all vectors within four different rare restriction sites at the 5'end and multiple cloning sites at the 3'end. In this way, a number of different fusion tags but also signal peptides can be incorporated at the N- and C-terminus of proteins, facilitating their expression, solubility and subsequent detection and purification. Fusion tags can be efficiently removed by treatment with site-specific peptidases, such as tobacco etch virus proteinase, thrombin, or sentrin specific peptidase 1, which leave only a few extra residues at the N-terminus of the protein. The combination of different expression systems in concert with the cloning approach in vectors that can fuse various tags makes the pCri System a valuable tool for high throughput studies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus subtilis / genetics
  • Carboxypeptidases A / chemistry
  • Carboxypeptidases A / genetics
  • Carboxypeptidases A / metabolism
  • Cloning, Molecular
  • Disulfides / chemistry
  • Escherichia coli / genetics
  • Genetic Vectors*
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / genetics
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / isolation & purification
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / metabolism
  • Pichia / genetics
  • Protein Engineering / methods*
  • Protein Processing, Post-Translational
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / genetics*
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Solubility

Substances

  • Disulfides
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • CPA3 protein, human
  • Carboxypeptidases A
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase

Grant support

This study was supported in part by grants from European, Spanish, and Catalan agencies (FP7-HEALTH-2010-261460 “Gums&Joints”; FP7-PEOPLE-2011-ITN-290246 “RAPID”; FP7-HEALTH-2012-306029-2 “TRIGGER”; BFU2012-32862; CSD2006-00015; and 2014SGR9). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.