The isolation of the cDNA sequence encoding the human neuronal kinesin (a force-generating motor protein which transports various membrane organelles along microtubules in an ATP-dependent manner) heavy chain (nKHC) and the construction of expression vectors to produce the full-length nKHC and its domains in Escherichia coli is described. By tuning up the conditions for the expression of nKHC, a sufficient amount of the soluble protein intragenously tagged with 6xHis tag was obtained and purified by nickel chromatography. The recombinant structural domains of nKHC, including the motor domain (FKHC1--amino acids 1-330), the microtubule binding domain (FKHC2--amino acids 174-315) and the coiled-coil stalk domain (FKHC3--amino acids 331-906) were used to determine the epitope location for monoclonal antibodies KN-01, KN-02, and IB II raised against different kinesin heavy chains. The antibodies were shown to recognize epitopes located in the stalk domain of nKHC and represent thus useful probes for this domain.

Institute of Microbiology Academy of Sciences of the Czech Republic 142 20 Prague Czechia

Zobrazit více v PubMed

Nature. 1970 Aug 15;227(5259):680-5 PubMed

Neuron. 1994 May;12(5):1059-72 PubMed

Folia Biol (Praha). 2002;48(2):77-9 PubMed

Folia Biol (Praha). 1998;44(1):35-6 PubMed

Nucleic Acids Res. 1988 Jul 11;16(13):6127-45 PubMed

Hybrid Hybridomics. 2002 Dec;21(6):457-62 PubMed

Methods Mol Biol. 2001;164:43-8 PubMed

Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4 PubMed

J Cell Biol. 1998 Nov 16;143(4):1053-66 PubMed