以大腸桿菌生合成grafted cyclotides做為抑制載脂蛋白E4區域交互作用之結構校正器

Abstract

Cyclotides是源自於植物的一種環狀胜肽，在結構上具有由三個分子內的雙硫鍵而形成胱胺酸紐結，此特徵造就了cyclotides結構上的穩定性。有許多的cyclotides，例如：McoTI-II和kalata B1，已經被發現具有細胞穿透的功能，同時在cyclotides的環狀區域上，對於胺基酸序列的置換擁有良好耐受性。這些特徵使得cyclotides具有潛力，作為以胜肽為骨架的藥物設計。在本篇研究中，以載脂蛋白E4上的第50到70號胺基酸序列取代McoTI-II與kalata B1的第6環狀區域上的胺基酸序列，藉此去抑制載脂蛋白E4上第61號位置精胺酸與第255號位置麩醯胺酸的結構區域交互作用。根據過去的研究顯示，cyclotides能透過大腸桿菌生合成的方法來製備，因此在本篇研究設計中，嘗試以大腸桿菌來表現改造過的McoTI-II與kalata B1，同時共同表現butelase環化酶來協助cyclotides的環化。Butelase能將受質蛋白的N端與C端進行分子內相連，因而協助cyclotides形成環狀結構。本實驗嘗試將butelase以及改造過的McoTI-II與kalata B1的基因編碼序列放入pET20b載體中表現，由於pET20b帶有pelB訊號序列，因此能將表現出的蛋白質運送至膜間質，以提供氧化環境去協助雙硫鍵之形成與氧化折疊。在本篇研究中，以BL21(DE3)、C41(DE3)與C43(DE3)pLysS作為表現目標蛋白的菌株。在C41(DE3)菌株中，初步以IPTG誘導蛋白質表現的結果顯示，pET20b-McoTI-IIa組別中在SDS-PAGE上有出現小於10 kDa的條帶，但透過LC-MS/MS分析的結果顯示沒有鑑定到目標蛋白。而在另一方面，無論有無pelB訊號序列的存在，目標蛋白質都沒有被過度表現。因此，是否改造過的cyclotides能影響載脂蛋白E4的結構域交互作用並沒有結論。

Cyclotides are cyclic peptides originated from plants possessing ultra-stability because of their intramolecular cystine knot involving three disulfide bonds and a cyclic structure. Several cyclotides such as Momordica cochinchinensis trypsin inhibitor-II (McoTI-II) and kalata B1 have been shown to display cell-penetrating properties and exhibit a great tolerance to amino acid substitutions in loop structure. These features make cyclotides potential scaffolds for peptide-based drug design. In this study, two cyclotides were designed to disrupt the R61-E255-mediated domain interaction in apolipoprotein E4 (apoE4) by replacing the loop 6 region of McoTI-II and kalata B1 with a fragment spanning amino acid residues 50 to 70 of apoE4. According to previous studies, cyclotides can be biosynthesized in E. coli for the preparation of genetically-encoded libraries. Here, these two cyclotides were intended to be generated through biosynthesis in E. coli with a cyclase, butelase. Butelase was used to connect the N and C termini of substrate proteins to assemble a cyclic structure. Coding sequences of grafted McoTI-II and kalata B1 were cloned into pET20b, an expression vector carrying a pelB signal sequence for directing the produced proteins to the periplasm, which provides an oxidative folding environment. In this study, bacterial strains, BL21(DE3), C41(DE3) and C43(DE3)pLysS was used to express target proteins. Preliminary results of protein expression in C41(DE3) strain showed a more intense band below 10 kDa was observed in the pET20b-McoTI-IIa group on SDS-PAGE analysis. However, no fragments of target protein was identified by LC-MS/MS analysis. On the other hand, whether or not pelB signal sequence was present, the target protein was not overexpressed after IPTG induction. Therefore, whether the grafted cyclotides can disrupt the domain interaction in apoE4 was still inconclusive.