Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 53, Issue 1, Pages 40-50Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2006.12.006
Keywords
ligation independent cloning; LIC; SUMO; subtractive purification; expressed protein ligation; EPL; structural genomics
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Funding
- NIGMS NIH HHS [GM64676, P01 GM064676] Funding Source: Medline
- NIMH NIH HHS [R01 MH073060, MH73060] Funding Source: Medline
- NINDS NIH HHS [R21 NS048548, R01 NS065140] Funding Source: Medline
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With demand increasing for the production of many different proteins for biophysical or biochemical analyses, rapid methods are needed for the cloning, expression and purification of native recombinant proteins. In particular, generic methods are required that are independent of the target gene sequence. To address this challenge we have constructed four Escherichia coli expression vectors that can be used for ligation independent cloning (LIC) of an amplified target gene sequence. These vectors represent the combinatorial pairing of two different parent vector backbones with two different affinity tags. The target gene is cloned downstream of the sequence coding for an affinity-tagged small ubiquitin related modifier (SUMO). Using enhanced green fluorescent protein (eGFP) as an example we demonstrate that the LIC procedure works with high efficiency for all four of the vectors. We also show that the resultant recombinant SUMO fusion proteins can be overexpressed in E coli and readily isolated by standard affinity purification techniques. Importantly, the purified fusion product can be treated with recombinant SUMO hydrolase to yield a mature target protein with any residue except proline at the amino terminus. We demonstrate an application of this by generating recombinant eGFP containing a non-native amino terminal cysteine residue and using it as a substrate for expressed protein ligation (EPL). The reagents and techniques described here represent a generic method for the rapid cloning and production of a target protein, and would be appropriate for a high throughput genomic scale expression project. (c) 2006 Elsevier Inc. All rights reserved.
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