New high-cloning-efficiency vectors for complementation studies and recombinant protein overproduction in Escherichia coli and Salmonella enterica

Plasmid. 2016 Jul;86:1-6. doi: 10.1016/j.plasmid.2016.05.001. Epub 2016 May 24.

Abstract

Galloway et al. recently described a method to alter vectors to include Type IIS restriction enzymes for high efficiency cloning. Utilizing this method, the multiple cloning sites of complementation and overexpression vectors commonly used in our laboratory were altered to contain recognition sequences of the Type IIS restriction enzyme, BspQI. Use of this enzyme increased the rate of cloning success to >97% efficiency. L(+)-Arabinose-inducible complementation vectors and overexpression vectors encoding N-terminal recombinant tobacco etch virus protease (rTEV)-cleavable H6-tags were altered to contain BspQI sites that allowed for cloning into all vectors using identical primer overhangs. Additionally, a vector used for directing the synthesis of proteins with a C-terminal, rTEV-cleavable H6-tag was engineered to contain BspQI sites, albeit with different overhangs from that of the previously mentioned vectors. Here we apply a method used to engineer cloning vectors to contain BspQI sites and the use of each vector in either in vivo complementation studies or in vitro protein purifications.

Keywords: Complementation vectors; Escherichia coli; High-efficiency cloning vectors; Overexpression vectors; S. enterica; Type IIS restriction cloning.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Base Sequence / genetics
  • Cloning, Molecular / methods*
  • Deoxyribonucleases, Type II Site-Specific / metabolism*
  • Endopeptidases / genetics*
  • Escherichia coli / genetics*
  • Genetic Vectors / genetics
  • Plasmids / genetics
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / metabolism
  • Salmonella enterica / genetics*

Substances

  • Recombinant Fusion Proteins
  • Deoxyribonucleases, Type II Site-Specific
  • Endopeptidases
  • TEV protease