麩胺酸異位酶S次單元(glutamate mutase S component, MutS)是麩胺酸異位酶中的鈷胺素結合區(cobalamin-binding domain)，共含131個胺基酸。 本論文嘗試建立以化學酵素法合成此蛋白。首先，將MutS分為兩個片段分別合成： 一為從N端算起至第25個胺基酸(peptide 1)，以Fmoc固相胜肽合成法生產，於第25個胺基酸之C末端(C-terminal)處理為硫酯(thioester)； 另一部分為第26胺基酸開始至131殘基(peptide 2)。 以傳統的基因重組法將第26個胺基酸絲胺酸(serine)更改為半胱胺酸(cysteine)，同時在第26胺基酸前插入一段具有專一性的菸草蝕刻病毒蛋白酵素(tobacco etch virus NIa protease, TEV protease) 的辨識位置(E-X-L-Y-X-N↓)，轉殖入大腸桿菌中大量表現，並以TEV蛋白酵素作為切割基因重組蛋白質(His-TEV-MutS26C)的工具。 經由菸草蝕刻病毒蛋白酵素水解處理過後的蛋白質則可因此生產出N末端為半胱胺酸(cysteine)的蛋白質片段，以作為接合(ligation)的物件之一。 將上述兩個片段以自然化學接合法(native chemical ligation)將peptide 1之C端的thioester與peptide 2之 N端的半胱胺酸在中性條件下發生化學選擇之硫酯轉移反應(chemoselective transthioesterification)，在接合的位置形成新的醯胺鍵(amide bond)成一完整的蛋白質。由於化學法合成胜肽的優點，是可以引入非天然型或D型的胺基酸於產物中，以彌補一般固相胜肽合成法合成胜肽的長度有所限制的缺點。 本論文嘗試利用自然化學接合法合成分子量為14 kd的MutS次單元。 Glutamate mutase S component (MutS) is the smallest known protein subunit that carries cobalamin-binding domain with molecular masses of 14,748 Da. The chemo-enzymatic synthesis of this small monomeric protein by the native chemical ligation method is the long-term goal of in this study. Two unprotected peptide segment, 1 and 2, were synthesized separately. The segment 1 (residue 1-25) was readily prepared in good yield by solid-phase peptide synthesis. A thio-ester group was added to the carboxyl end of the segment 1. The segment 2 (residue 26-131) was produced by the over-expression of an engineered and truncated mutS gene. After treatment with TEV protease to remove the hexa-His purification tag, the segment 2 peptide carries a cysteine group at its N-terminal end. The ligation of above two fragments a rapid intramolecular S→N acyl shift through will be subsequently carried out in our group. This result indicates that incorporation of an artificial amino acid residue into the conserved cobalamin-binding motif is feasible by using this synthetic strategy.
