Construction of a dual-tag system for gene expression, protein affinity purification and fusion protein processing |
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Authors: | Hassan Motejadded Josef Altenbuchner |
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Institution: | 1. Institut für Industrielle Genetik, Universit?t Stuttgart, Allmandring 31, 70569, Stuttgart, Germany
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Abstract: | An E. coli vector system was constructed which allows the expression of fusion genes via a l-rhamnose-inducible promotor. The corresponding fusion proteins consist of the maltose-binding protein and a His-tag sequence
for affinity purification, the Saccharomyces cerevisiae Smt3 protein for protein processing by proteolytic cleavage and the protein of interest. The Smt3 gene was codon-optimized
for expression in E. coli. In a second rhamnose-inducible vector, the S. cerevisiae Ulp1 protease gene for processing Smt3 fusion proteins was fused in the same way to maltose-binding protein and His-tag sequence
but without the Smt3 gene. The enhanced green fluorescent protein (eGFP) was used as reporter and protein of interest. Both
fusion proteins (MalE-6xHis-Smt3-eGFP and MalE-6xHis-Ulp1) were efficiently produced in E. coli and separately purified by amylose resin. After proteolytic cleavage the products were applied to a Ni-NTA column to remove
protease and tags. Pure eGFP protein was obtained in the flow-through of the column in a yield of around 35% of the crude
cell extract. |
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Keywords: | Gene expression Protein affinity purification Proteolytic cleavage Ulp1 protease SUMO protein |
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