Construction of recombinant rabbit uterine smooth muscle myosin light chain kinase cDNA for expression in insect cells. Satishkumar Noonepalle

ISBN: 9781109063356

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109 pages


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Construction of recombinant rabbit uterine smooth muscle myosin light chain kinase cDNA for expression in insect cells.  by  Satishkumar Noonepalle

Construction of recombinant rabbit uterine smooth muscle myosin light chain kinase cDNA for expression in insect cells. by Satishkumar Noonepalle
| NOOKstudy eTextbook | PDF, EPUB, FB2, DjVu, audiobook, mp3, ZIP | 109 pages | ISBN: 9781109063356 | 8.71 Mb

Myosin light chain kinases (MLCKs) are a group of enzymes that catalyze the transfer of gamma-phosphate from MgATP to a serine residue near the N-terminus of myosin regulatory light chain (RLC) (Kamm and Stull 2001). Smooth muscle MLCK (smMLCK) isMoreMyosin light chain kinases (MLCKs) are a group of enzymes that catalyze the transfer of gamma-phosphate from MgATP to a serine residue near the N-terminus of myosin regulatory light chain (RLC) (Kamm and Stull 2001).

Smooth muscle MLCK (smMLCK) is expressed in all adult tissues including skeletal and non-muscle tissues (Kamm and Stull, 2001) where smMLCK phosphorylates only smooth muscle RLC as its substrate (Leachman et al., 1992). For a better understanding of the mechanism of action of smMLCK, chimeric MLCKs were previously constructed in our lab by Pondugula (2004) and stable cell lines expressing wild type and chimeric proteins were established by Kandlur (2006).

Recently, sequencing of the smMLCK clone in pCMV-5 we obtained from Dr. J. T. Stull (UT Southwestern Medical Center, Dallas, TX) which was used for all chimera constructs, revealed unwanted mutations and deletions in the 5 terminus. Therefore, construction of the wild type rabbit uterine smooth muscle MLCK expression vector has become essential to reconstruct the chimeras by domain swapping. The goal of this research was to construct recombinant rabbit uterine smooth muscle myosin light chain kinase (smMLCK) cDNA from total RNA isolated from frozen rabbit uterine muscle for expression in an insect cell host system.

The construct was verified for expression of rabbit uterine myosin light chain kinase protein by performing transient transfection into the High Five(TM) insect cell expression system (Invitrogen(TM)) and western blot analysis using a monoclonal anti MLCK anti body. Expressed protein has a C-terminus his-tag facilitating easy one step purification by nickel affinity chromatography.



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