| Summary: | 碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 93 === Sucrose phosphate synthase (SPS) is an important regulatory enzyme of sucorse biogenic reaction in plants. The purpose of this study is to express sucrose phosphate synthase from shoots of Bamboo (BOSPS) by eukaryotic expression system with posttranslational modification.
At first, forward (with restriction site NotⅠ) and reverse (with restriction site ApaⅠ) primers were designed according to the both terminal sequences of BOSPS cDNA. Then, BOSPS cDNA containing these restriction sites was amplified by PCR and cloned into T vector (yT&A). Both of the recombinant plasmid pTBOSPS and pPICZ A vector were digested by restriction enzymes ApaⅠand NotⅠ sequentially. Therefore, BOSPS cDNA can be cloned into pPICZ A vector (pPBOSPS) by the terminal complementary cohesive end. Being confirmed the correct nucleotide sequence by sequencing, recombinant plasmid pPBOSPS could be linearized and transformed into Pichia pastoris (X-33).
Before scale-up of BOSPS expression induced by the addition of methanol, determining the Mut phenotype and optimal time post-induction to harvest. Finally, expressed BOSPS was purified by a procedure involving precipitation with 10~30% PEG and affinity chromatography, and analyzed for its characterization.
|
|---|
