Human kinesins and methods of producing and purifying human kinesins

Abstract

Described herein are methods of producing kinesins. In a preferred embodiment, the kinesins are produced from a prokaryote, most preferably, a bacterial cell. Bacterial expression offers several advantages over systems previously utilized, such as, for example, Bacculovirus. The yield of protein is higher, the cost of the expression setup is lower, and creation of alternative expression vectors is easier. The concern of copurifying a contaminating activity from the expression host is also eliminated since bacteria, in contrast to the bacculovirus expresion system, do not have kinesin like proteins. Also described herein are purified kinesins, preferably unglycosylated and methods of use.

We claim:  
     
         1 . A method of producing a human mitotic kinesin protein excluding Kid comprising a motor domain comprising: 
 expressing a nucleic acid comprising a nucleic acid encoding a human mitotic kinesin protein comprising a motor domain in a bacterial cell and    substantially purifying said human mitotic kinesin protein.    
     
     
         2 . A method of producing a human kinesin protein comprising a motor domain comprising: 
 expressing a nucleic acid comprising a nucleic acid encoding a human kinesin protein comprising a motor domain in a bacterial cell and    substantially purifying said human kinesin protein, wherein said kinesin protein is selected from the group consisting of chromokinesin, KSP, CENP-E, MKLP1, HSET, Kif15, Kin2, Kif1A and MCAK.    
     
     
         3 . A method of producing a human kinesin protein comprising a motor domain and at least three epitope tags comprising: 
 expressing a nucleic acid encoding a human kinesin protein comprising a motor domain in a bacterial cell and    substantially purifying said human kinesin protein.    
     
     
         4 . The method of  claim 3  wherein KHC is excluded.  
     
     
         5 . A method of producing a kinesin protein comprising a motor domain comprising: 
 expressing a nucleic acid encoding a kinesin protein comprising a motor domain in a bacterial cell and    substantially purifying said kinesin protein, wherein said kinesin is selected from the group consisting of Kin2, chromokinesin, HSET, Kif15, Kif1A, BimC and MKLP1.    
     
     
         6 . A substantially pure unglycosylated human mitotic kinesin protein excluding kid comprising a motor domain.  
     
     
         7 . A substantially pure unglycosylated human mitotic kinesin protein, wherein said kinesin protein is selected from the group consisting of chromokinesin, KSP, CENP-E, MKLP1, HSET, Kif15, Kin2, Kif1A and MCAK.  
     
     
         8 . A substantially pure unglycosylated human kinesin protein comprising a motor domain and at least three epitope tags.  
     
     
         9 . A substantially pure unglycosylated kinesin protein comprising a motor domain, wherein said kinesin is selected from the group consisting of Kin2, chromokinesin, BimC, HSET, Kif15, Kif1A and MKLP1.  
     
     
         10 . A bacterial cell comprising a nucleic acid comprising a nucleic acid encoding a kinesin selected from the group consisting of chromokinesin, BimC, HSET, Kif15, Kin2, and Kif1A.  
     
     
         11 . A bacterial cell comprising a nucleic acid comprising a nucleic acid encoding a human kinesin selected from the group consisting of chromokinesin, Kin2, BimC, Kif1A, KSP, CENP-E, MCAK, HSET and Kif15.  
     
     
         12 . An assay to identify a candidate agent which binds to a kinesin protein of  claim 6 ,  7 ,  8 , or  9  comprising: 
 combining a candidate agent with a kinesin protein of  claim 6 ,  7 ,  8 , or  9  and determining whether binding occurs.  
 
     
     
         13 . An assay to identify a modulator of a kinesin protein of  claim 6 ,  7 ,  8 , or  9  comprising: 
 detecting activity of a kinesin protein of  claim 6 ,  7 ,  8 , or  9  in the presence and absence of a candidate agent wherein a change in activity indicates a modulator.  
 
     
     
         14 . The assay of  claim 13  wherein said activity is ATPase activity and/or microtubule binding or gliding.  
     
     
         15 . A substantially purified unglycosylated peptide selected from the group consisting of K335, Q475, D679, FL1, P166, H195, FL2, E433, R494, E658, L360, K491, S553, M329, T340, S405, V465, T488, M1, M2, M3, M4, M5, M6, FL3, A2N370, A2M511, K519, E152.2, Q151.2, Q353 and M472.