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.
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