Construction of pBluescript KS+-hPLK

1.25Kb hPLK amplified by RT-PCR with primer P220 and P221
                                               ↓       
Cloned into EcoR V site of pT7Blue by TA cloning
                                               ↓ 
wild type hPLK sequence is obtained by PCR based site-directed mutagenesis to
correct the errors in the sequence.
(P239/P240 correct G31.P241/P242 correct T173 to C and G179 to A)
                                               ↓ 
two mutant hPLK are subsequently made from the wild type 1.25Kb fragment:
K82D:P235/P236 change bp 245.K82M hPLK has no kinase activity.
         AAG for lysine 82 is changed to ATG for methionine.
T210D:P245/P246 change bp A627-C628 into G627-A628.
         T210D hPLK has increased kinase activity.
         Threonine(ACC)210 is changed to Aspartic acid(GAC)
                  
pT7Blue-hPLK WT, K82M,and T210D cut by BamH I
                                               ↓  
hPLK BamH I fragment is purified by electoelution  →    product 1   

W2C clone(partial hPLK sequence between a.a. 258 to stop codon, in pGAD•GH) 
is cut by BamH I/EcoR I
                                               ↓    
hPLK BamH I/EcoRI fragment is purified by gel electroelution 
                                               ↓ 
The W2 fragment is cloned into pBluescript KS+ to form pBlueScript KS+-W2C
                                               ↓ 
A    mutant is made from pBlue KS+-W2C:
△KWVD: remove sequence  between bp 1237 and bp 1249.
               The deletion remove K413-W414-V415-D416. These four amino acids are
    part of consensus polo box sequence.△KWVD should disrupt the location of hPLK. 


                   

pBlue KS+-W2C (WT) and △KWVD 
are  cut  with BamH I, treated with cip → product 2    

hPLK BamH I fragment is purified by electoelution (product 1)
                                     +
pBlue KS+-W2C (WT) and 
△KWVD are  cut  with BamH I, treated with cip (product 2 )
                                    ↓
complete hPLK sequence in pBluescript KS+
Four construction:WT, K82M, T210D, and △KWVD 



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