Cloning, characterization and expression of A. thaliana G protein α-subunit gene for structural studies
Bakkal, Süphan Emine (2003) Cloning, characterization and expression of A. thaliana G protein α-subunit gene for structural studies. [Thesis]
A combined PCR-cloning strategy was implemented to express GPAl for structural studies. To begin with the presence of GPAl, which İs the ?-subunit of heterotrimeric G proteins from Arabidopsis thaliana, was checked with restriction enzyme digestion and sequence analysis. PCR was done on the pCIT857 vector including the coding sequence of GPAl. Appropriate primers were designed with and without the restriction enzyme sites to clone the GPAl into both subcloning and expression vectors. Once the above objective was analysed, three different parallel approaches were used to clone-GPAl from the pCIT857 vector into the expression vectors. In the firstapproach, GPAl was subcloned into several vectors including pGEM® -T Easy (Promega), pCR® II- TOPO (Invitrogen), pCR® -XL-TOPO (Invitrogen) vectors. Then GPAl derived from these vectors were cloned into expression vectors containing PGEX-4T2 (Amersham Pharmacia), pGFPuv (Clonetech), pETM-11 and pETM-30 (EMBL, Heidelberg). In the second approach, GPAl from the pCIT857 vector was directly cloned into the expression vectors. Different fusion partners of expressed GPAl, GST in pGEX-4T2 and GFPuv in pGFPuv vector and His-taq in pETM vectors, were used to yield the most efficient expression of GPAl, that would form the basis of subsequent structural characterisation experiments.In the third approach, GPAl from pCIT was directly cloned into the expression vectors including pCR® T7/NT TOPO (Invitrogen) and pTrcHis® TOPO (Invitrogen) vectors. Of the three approaches, this one proved most convenient as the number of steps and manipulations was limited in comparisons to those above. During all subcloning and cloning steps, different strains of E. coli including XLlBlue, TOP10 and TOP10 F' were used as hosts for subcloning and BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS and BL21-CodonPlus® (DE3)-RIL were used for expression. The rationale being that different strains often perform differently.To summarise GPAl was successfully cloned into 3'MCS of pGFPuv, pCR® T7/NT TOPO (Invitrogen) and pTrcHis® TOPO (Invitrogen) vectors. In particular the verification was based upon the restriction enzyme digestion and sequencing data. Also the recombinant protein was expressed both using different IPTG concentrations and temperatures. But the expression was not detected by SDS-PAGE analysis. Recombinant protein is currently being expressed and further work is in progress to improve the properties of bacterial expression system.
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