Construction Of Full Length Recombinant And Calmodulin-binding Site Mutant Of Myosin Light Chain Kinase

Objective：Myosin light chain kinase （MLCK） is an important enzyme for smoothmuscle contraction. The intracellular concentration of Ca2+is increased throughstimulation, causing Ca2+to bind to calmodulin （CaM）. The complex of Ca2+and CaMactivates MLCK, which phosphorylates20-kDa myosin light chain （MLC20） to activatethe myosin ATPase activity. The activated myosin interacts with actin to induce smoothmuscle contraction. This is a classical pathway to regulate contraction. Besides itskinase activity, MLCK also has non-kinase activity which binds actin and myosin. Inaddition, it was reported that MLCK also plays an important role on smooth musclecontraction and the cytoskeleton recombinant. In order to research the effect ofnon-kinase activity of MLCK on the cytoskeleton recombinant, we engineered wildtype MLCK recombinant which can be expressed in eukaryotic cells by polymerasechain reaction （PCR） and molecular cloning technology. On this basis, we engineeredCaM-binding site mutant that was devoid of kinase activity by site-directed mutagenesisin1002-1022amino acid. The two constructions will be basis to further research thenon-kinase activity of MLCK on smooth muscle contraction.Methods：（1） Designed two primers containing NheⅠsite and Flag tag（DYKDDDDK） sequence through prokaryotic expression vector pCold-MLCK, andobtained a533bp gene by PCR reaction. Making use of restriction enzymeNhe Ⅰ/KpnⅠ digests the533bp gene and pCold-MLCK plasmid, we obtained insertfragment and vector fragment respectively. The insert fragment and vector fragmentwere ligated by T4DNA ligase and transformed into E.coli DH5α competent cells. Theexpression gene was the pcDNA-F-MLCK144plasmid we needed. Then thepCold-MLCK plasmid and pcDNA-F-MLCK144plasmid were digested by usingrestriction enzyme Kpn Ⅰ/EcoR Ⅰin order to obtain the3.4kb insert fragment and5.5kb vector fragment. After ligating insert and vector fragment, the full length MLCK genenplasmid pcDNA-F-MLCK was constructed.（2）According to CaM-binding domain,designed PCR primer containing NheⅠand EcoRⅠsite sequence. Taking the plasmidpcDNA-F-MLCK as the template, we obtained a700bp gene by PCR reaction. Makinguse of restriction enzyme Nhe Ⅰ/EcoR Ⅰ todigest PCR product and pcDNA3.1plasmid,we obtained insert fragment and vector fragment. After ligating the subclone plasmid ofpcDNA-MLCK700was obtained. According to CaM-binding domain, designed PCRprimer to transfer T⁹3016)、G3017to G³⁰¹⁶、C3017. Taking the subclone plamid as thetemplate, site-directed mutagenesis by PCR to recive a subclone mutantpcDNA-MLCK700-CaM. The insert fragment and vector fragment which were digestedfrom subclone plasmid and pCold-MLCK plasmid respectively by restriction enzymeNcoⅠ, and ligated, we got prokaryotic expression vector pCold-M-CaM.（3）Finally todigest the pCold-M-CaM plasmid and pcDNA-F-MLCK by Kpn Ⅰa nd EcoRⅠ, the3.4kb insert fragment including CaM-binding mutation site and5.5kb vector fragmentwere seperated. Bying ligating we obtained eukaryotic expression vector（pcDNA-F-M-CaM）.Results：（1）Taking the plasmid pCold-MLCK as the template, we obtained a533bp gene by PCR reaction. Making use of restriction enzyme NheⅠ/KpnⅠdigeststhe533bp gene and pCold-MLCK plasmid, we obtained insert fragment and vectorfragment respectively. The insert fragment and vector fragment were ligated by T4DNA ligase and transformed into E.coli DH5α competent cells. The expression genewas the pcDNA-F-MLCK144plasmid we needed. Then the pCold-MLCK plasmid andpcDNA-F-MLCK144plasmid were digested by using restriction enzymeKpnⅠ/EcoRⅠin order to obtain the3.4kb insert fragment and5.5kb vector fragment.After ligating insert and vector fragment, the full length MLCK genen plasmidpcDNA-F-MLCK was constructed.（2）The insert fragment containing CaM-bindingdomain with NheⅠ/EcoRⅠsite was amplified from pCold-MLCK. Making use ofrestriction enzyme NheⅠ/EcoRⅠto digest PCR product and pcDNA3.1plasmid, weobtained insert fragment and vector fragment. After ligating the subclone plasmid ofpcDNA-MLCK700was obtained. Taking the subclone plamid as the template,site-directed mutagenesis by PCR to recive a subclone mutant pcDNA-MLCK700-CaM.The insert fragment and vector fragment which were digested from subclone plasmidand pCold-MLCK plasmid respectively by restriction enzyme NcoⅠ, and ligated, wegot prokaryotic expression vector pCold-M-CaM.（3） Finally to digest the pCold-M-CaM plasmid and pcDNA-F-MLCK by KpnⅠand EcoRⅠ, the3.4kb insertfragment including CaM-binding mutation site and5.5kb vector fragment wereseperated. Bying ligating we obtained eukaryotic expression vector（pcDNA-F-M-CaM）.Conclusions：（1）In this study, we successfully constructed full length MLCKrecombinant pcDNA-F-MLCK.（2） We obtained CaM-binding site mutantpCold-M-CaM by using site-directed mutagenesis methods. And this mutant can beused to expression in prokaryocyte to study.（3）We obtained CaM-binding site mutantpcDNA--M-CaM by using PCR and molecular cloning technique. And this mutant canbe used to expression in eukaryocyte to study. This study provides the certain rationalefor further research on the molecular mechanism of smooth muscle contraction.