建立與評估具胞外分泌特性之TA表現載體

Abstract

本研究旨為建立一TA選殖與具胞外分泌能力之表現載體，以做為隨機突變研究之用，並對該載體進行初步之評估。 為建立此表現載體，我們首先於yT&A載體上插入一49 mer寡核苷酸，其中包含兩個重要的AhdI限制酶切位，此兩個切位為構成TA載體之要素，其為使建構完成之TA載體以AhdI切解後，於3’端處多一個T。並於此兩AhdI切位間接入一2000 bp之片段，以便於日後純化TA載體片段時，在DNA電泳膠片上可清楚辨識。為建構成具有胞外分泌特性之表現載體，我們再將含AhdI限制酶切位之片段搬移至pRSET A載體，並導入一訊息胜肽序列，以期利用pRSET A載體上之核醣體鍵結位置(ribosome binding site, rbs)以製造蛋白質。最後，再將整段建構之序列包含rbs、訊息胜肽序列及兩AhdI限制酶切位搬遷至pUC19載體上，以利用pUC19上之LacZ進行調控。 將Serratia marcescens之成熟幾丁質酵素基因重建在含有訊息胜肽之pUC19的TA載體(名為pUC _ TA _ sp)上，並以大腸桿菌XL1-blue進行表現，結果發現可成功地表現並分泌至胞外；另外，也將Bacillus circulans MH-K1幾丁聚醣酵素的基因(cns)建立於pUC _ TA _ sp載體中，以XL1-blue表現之，相同地，亦可在胞外發現明顯的活性，其酵素催化產物與本源(Native)酵素者相同，由活性與蛋白質分析顯示，於大腸桿菌胞內亦可發現酵素之活性。雖然此表現載體之胞外分泌能力雖尚未臻完美，但一具胞外分泌能力的TA表現載體已經建構完成。 此外，我們亦以error-prone PCR之方法，針對幾丁聚醣酵素基因進行隨機突變。並利用建構完成之含有訊息胜肽之pRSET A之TA載體(pRSET _ TA _ sp)進行接合，並以BL21(DE3)表現之。雖然，尚未由突變之基因庫中篩選出具特殊催化活性之幾丁聚醣水解酵素突變株，但已初步證實可利用此等TA表現載體(pRSET _ TA _ sp或pUC19 _ TA _ sp)進行隨機突變研究。

Constructing a TA cloning and expression vector with extracellular secretion feature is the main theme of this study. In order to create the TA site, two 49-mer oligonucleotides with the first 48-mer complementary to each other and an un-paired A remaining at the 3’ of each strand. Two AhdI sites and an XhoI site, in between the two AhdI sites, were designed in the 49-mer fragment. First of all, the 49-mer was inserted in the TA site of yT&A cloning vector. For the ease of purifying the digested TA vector for further application, a 2000 base-pair DNA fragment was incorporated in the XhoI site of the 49-mer to give a inserted AhdI-fragment. The AhdI-fragment was then reconstructed into pRSET A with a pre-existing signal peptide sequence derived from the ChiA of Serratia marcescens to give the pRSET _ TA _ sp vector. For constructing the expression vector, the DNA fragment from pRSET _ TA _ sp containing rbs (ribosome binding site), signal peptide sequence, and the AhdI-fragment was constructed in pUC19. LacZ promoter of pUC19 will then be used for induction. The resulting vector was named as pUC _ TA _ sp and should be ready for expression application. Both the chitinase (chiA) from S. marcescens and the chitosanase (cns) from Bacillus circulans MH-K1 were inserted in the pUC _ TA _ sp vector for enzyme secretion analysis. Result showed that secretion property of the pUC _ TA _ sp was quite significant. Enzymatic products of the two recombinant proteins were virtually identical to those of the native enzymes. A random mutagenesis study based on the error-prone PCR technique was performed by using pRSET _ TA _ sp as screening vector. Though no mutant with particular interest was obtained from the mutagenic library, the TA vector has been successfully constructed.