Cloning, expression, purification and refolding of microtubule affinity-regulating kinase 4 expressed in Escherichia coli.
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.
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110025, India.
Farha
F
Naz
Mohd
M
Asad
Pawan
P
Malhotra
Asimul
A
Islam
Faizan
F
Ahmad
Md Imtaiyaz
MI
Hassan
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D007700:Kinetics; D008870:Microtubules / Q000737:chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012521:Sarcosine / Q000031:analogs & derivatives / Q000737:chemistry
D011994:Recombinant Proteins; C025231:sarkosyl; D000255:Adenosine Triphosphate; D048049:Extracellular Signal-Regulated MAP Kinases; D000251:Adenosine Triphosphatases; D012521:Sarcosine