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Current Topics in Peptide & Protein Research   Volumes    Volume 3 
Use of an engineered His-tag to improve the purification of recombinant wild-type and modified pig heart cytosolic malate dehydrogenase
Francisca Trejo, Josep Lluís Gelpí, Monterrat Busquets, Antoni Cortés
Pages: 173 - 180
Number of pages: 8
Current Topics in Peptide & Protein Research
Volume 3 

Copyright © 1999 Research Trends. All rights reserved


The complete cDNA that encodes pig cytosolic malate dehydrogenase was obtained from a pig cDNA library by PCR techniques. The cloned cDNA was expressed in TG2 Escherichia coli cells at concentrations of up to 30 mg/1 of culture. Conventional purification techniques give homogeneous enzyme after 4-5 days work with an overall yield of 54%. To shorten the process and increase the yield we assayed the use of an engineered His-tag at the C-terminus of the protein, in combination with affinity chromatography through HiTrap Chelating-Sepharose. The nature of the immobilized metal and the conditions of elution were analyzed. The resulting optimal conditions, using Ni2+as cation and an imidazole gradient for elution give an overall yield above 90% in about 2 h with a purity similar to that obtained by conventional procedures. The optimized method was applied to several mutant sequences with varying stability, with equivalent results. Characterization of the modified malate dehydrogenase (kinetic properties, enzyme stability, coenzyme affinity) revealed that the engineered His-tag has no influence in the overall behaviour of the enzyme.

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