Investigation of cloning strategies for A. thaliana G Protein [alpha]-subunit gene in Pichia Pastoris

Official URL: http://risc01.sabanciuniv.edu/record=b1124809 (Table of Contents)

In this thesis a strategy was developed to clone and express the gene of the A. thaliana heterotrimeric G-protein [alpha] subunit (GPA1). For this purpose an appropriate eukaryotic expression system was chosen to produce large quantities of high purity recombinant protein. GPA1 was amplified by PCR and cloned using a Pichia pastoris expression system. Two different plasmids pPICZC+GPA1 and pPICZ[alpha]B+GPA1' were constructed. pPICZC+GPA1 was designed for intracellular expression whereas pPICZ[alpha]B+GPA1' contained a signal peptide facilitating secretion of the recombinant protein into the extracellular medium. The possibility of using different yeast strains that may improve expression was explored. Recombinant synthesis of GPA1 was achieved with the pPICZC+GPA1 construct using the strain GS115, which shows Mut[+] phenotype. Expression was followed by monitoring growth of yeast as well as western blots of cellular extracts at different time points during induction. This study describes the first report of expression of A. thaliana GPA1 gene in a eukaryotic system and constitutes a critical step forward in studies of G-proteins in plants. It follows to reason that the availability of purified recombinant GPA1 will enable biochemical characterization, comparison with its mammalian counterparts and facilitate structural studies.