Design and establishment of a vector system that enables production of multifusion proteins and easy purification by a two-step affinity chromatography approach

J Microbiol Methods. 2014 Oct;105:47-50. doi: 10.1016/j.mimet.2014.07.008. Epub 2014 Jul 13.


The LE (LguI/Eco81I)-cloning procedure allows a step-wise, directional fusion of multiple DNA-fragments into a vector by utilizing two restriction enzymes generating identical non-palindromic overhangs. This strategy was applied to produce heat-stable cellulase-fusion proteins containing up to five single moieties. Terminal affinity tags enable efficient purification using a simple two-step approach.

Keywords: Gene fusion; Heat-stable cellulases; Multiple fusion approach; Two-step affinity chromatography; Type IIS restriction enzyme.

Publication types

  • Evaluation Study

MeSH terms

  • Cellulose / genetics
  • Cellulose / isolation & purification
  • Chromatography, Affinity / methods*
  • Protein Engineering / methods*
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / isolation & purification*


  • Recombinant Fusion Proteins
  • Cellulose