綠竹筍蔗糖磷酯合成酶在酵母菌中之表現

Abstract

蔗糖磷酯合成酶 (sucrose phosphate synthase，簡稱SPS) 為高等植物調控蔗糖生合成之重要酵素。本論文目的，即希望以具真核細胞轉譯後修飾作用之表現系統，表現綠竹筍蔗糖磷酯合成酶(BOSPS)。 首先，依據BOSPS cDNA 之5‘及3’ 端序列，設計含特定限制酶 (NotⅠ及ApaⅠ) 切位之引子，利用聚合酶鏈鎖反應 (PCR)，即得兩端分別具 NotⅠ 及 ApaⅠ 切位之BOSPS cDNA；將此cDNA 接入T載體yT&A，轉形至大腸桿菌 (JM109) 中保存。續以限制酶NotⅠ及ApaⅠ將其自T載體切下，與經相同限制酶處理之pPICZ A表現載體接合(pPBOSPS)後，轉形至大腸桿菌 (JM109) 進行定序分析；待序列確認無誤，以限制酶 SacⅠ將此重組表現載體切成線狀，轉形至Pichia pastoris 中 (X-33)。 確定酶母菌轉形株表現型及最適表現時間點後，即可以甲醇誘導BOSPS大量表現；繼則以10~30% PEG沈澱及鎳離子親和層析管柱純化之，所得酵素則可分別進行各項生化性質分析。

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.