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Cloning, Expression, Purification and Refolding of Microtubule Affinity-Regulating Kinase 4 Expressed in Escherichia coli

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Abstract

Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) is a member of the family Ser/Thr kinase and involved in numerous biological functions including microtubule bundle formation, nervous system development, positive regulation of programmed cell death, cell cycle control, cell polarity determination, cell shape alterations, cell division etc. For various biophysical and structural studies, we need this protein in adequate quantity. In this paper, we report a novel cloning strategy for MARK4. We have cloned MARK4 catalytic domain including 59 N-terminal extra residues with unknown function and catalytic domain alone in PQE30 vector. The recombinant MARK4 was expressed in the inclusion bodies in M15 cells. The inclusion bodies were solubilized effectively with 1.5 % N-lauroylsarcosine in alkaline buffer and subsequently purified using Ni–NTA affinity chromatography in a single step with high purity and good concentration. Purity of protein was checked on sodium dodecyl sulphate–polyacrylamide gel electrophoresis and identified by using mass spectrometry immunoblotting. Refolding of the recombinant protein was validated by ATPase assay. Our purification procedure is quick, simple and produces adequate quantity of proteins with high purity in a limited step.

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Acknowledgments

FN acknowledges the Council of Scientific and Industrial Research for the award of fellowship. We sincerely thank Harvard Medical School for providing MARK4 cDNA. FA and MIH are thankful to the Department of Science and Technology for funding.

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Correspondence to Md Imtaiyaz Hassan.

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Naz, F., Asad, M., Malhotra, P. et al. Cloning, Expression, Purification and Refolding of Microtubule Affinity-Regulating Kinase 4 Expressed in Escherichia coli . Appl Biochem Biotechnol 172, 2838–2848 (2014). https://doi.org/10.1007/s12010-014-0733-5

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  • DOI: https://doi.org/10.1007/s12010-014-0733-5

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