A versatile ligation-independent cloning method suitable for high-throughput expression screening applications

Nucleic Acids Res. 2007;35(6):e45. doi: 10.1093/nar/gkm047. Epub 2007 Feb 22.


This article describes the construction of a set of versatile expression vectors based on the In-Fusion cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His(6)-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Cell Line
  • Cloning, Molecular / methods*
  • Escherichia coli / genetics
  • Genes, Viral
  • Genetic Vectors / chemistry
  • Humans
  • Neisseria / genetics
  • Nerve Tissue Proteins / biosynthesis
  • Nerve Tissue Proteins / genetics
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Transcription Factors / genetics


  • Bacterial Proteins
  • KIAA0319 protein, human
  • Nerve Tissue Proteins
  • Recombinant Fusion Proteins
  • Transcription Factors