中華眼鏡蛇（Naja atra）蛇毒中，有高達約百分之八十之蛇毒為富含雙硫鍵（disulfide-rich）的毒蛋白。其中，又以致命性的短鏈神經毒素（short chain α-neurotoxins）及心臟毒素（cardiotoxins）兩類三指型毒素為大宗。在大腸桿菌中製備這樣富含雙硫鍵的重組蛋白，同時具有結構及活性，是長久以來科學家欲解決的問題；因大腸桿菌細胞質環境不利於富含雙硫鍵的蛋白進行正確的結構折疊，而產生之重組蛋白也可能造成其死亡。此篇論文中，我們欲應用雙硫鍵異構酶（Disulfide bond C, DsbC）接合之重組蛇毒蛋白，預期雙硫鍵異構酶能在表達系統中，幫助重組蛇毒蛋白進行正確結構折疊。在分別表現飯匙倩短鏈神經毒素及其中一心臟毒素（Cardiotoxin III）後，我們發現透過這樣的重組蛋白表現策略，可以穩定生產具有結構的重組蛇毒蛋白。從二維核磁共振光譜ˊ中可以看出，短鏈神經毒素及心臟毒素之重組蛋白質與天然蛋白質光譜具有相似性，顯示此表現系統能夠有效產生與天然結構相似的重組蛋白。迄今，蛇毒咬傷之治療方式並不完備，某些案例中，抗蛇毒血清無法完全中和蛇毒毒性。因此，在大腸桿菌中成功表達重組蛇毒蛋白，不僅可以提供穩定及結構正確的三指型蛇毒蛋白作為新型免疫原之開發，未來結合致突變作用，還能進一步利用核磁共振光譜，找出毒蛋白之關鍵胺基酸，解答毒素之生理機制。

Disulfide-rich proteins account for about 80% of snake venom. Among the proteins, neurotoxins (NTXs) and cardiotoxins (CTXs) are most dominant. The preparation of the recombinant disulfide-rich proteins in E. coli expression system is a long-standing problem because E. coli cytoplasm is not proper for the protein folding and the possible protein toxicity could cause cell lysis. To prepare functional active NTXs and CTXs, we developed a strategy by fusing a unique protein partner, disulfide bond isomerase (Disulfide bond C, DsbC) at the N-terminus of the target proteins. DsbC assists protein folding by converting aberrant disulfide bonds to correct formation in the periplasm. Here, we tested on short chain α-neurotoxin (sNTX) and Cardiotoxin III (CTXA3) from Naja atra. Through the strategy, sustainable amounts of NTXs and CTXs could be prepared from E. coli expression system. NMR method validated the proper protein folding that HSQC and 1H-1H TOCSY demonstrated spectral similarity between native and recombinant proteins. Subsequently, 13C and 15N-labeled proteins are obtained for NMR protein backbone assignment. To date, to cure snakebite poisoning, the implementing of correct antivenom is still the most effective treatment. However, in many cases, the toxicity couldn’t be fully neutralized even the corresponding antibody in the antivenom demonstrated great affinity to the toxic molecules. To realize the structural explanation, NMR shows potential to differentiate the residues interacting with the antibody. The study could put emphasis on epitope determination and provide a new prospective for the development of new generation of antivenom.

Contents
中文摘要 i
Abstract iii
Acknowledgement iv
Abbreviation vi
1 Introduction - 1 -
2 Materials and methods - 7 -
2.1 Native protein purification - 7 -
2.1.1 FPLC purification of MMW from crude venom - 7 -
2.1.2 HPLC purification of native sNTX and CTXA3 - 7 -
2.1.3 NMR sample preparation for native proteins - 7 -
2.2 Recombinant sNTX and CTXA3 expression - 8 -
2.2.1 Construct of rsNTX and rCTXA3 - 8 -
2.2.2 Recombinant DsbC-sNTX expression - 8 -
2.2.3 Recombinant DsbC-CTXA3 expression - 9 -
2.2.4 Purification of DsbC-rsNTX and DsbC-rCTXA3 - 9 -
2.2.5 TEV protease preparation - 11 -
2.2.6 NMR sample preparation for rsNTX and rCTXA3 - 11 -
2.3 Quantification of proteins - 11 -
2.4 Mass spectroscopy - 11 -
2.5 Recombinant sNTX HSQC and backbone assignment - 12 -
3 Results - 21 -
3.1 Native protein purification - 21 -
3.1.1 MMW FPLC and HPLC profiles - 21 -
3.2 Characterization of native protein by NMR - 21 -
3.2.1 2D 1H-1H TOCSY of sNTX and CTXA3 - 21 -
3.2.2 2D 1H-15N HSQC of sNTX and CTXA3 - 22 -
3.3 Recombinant sNTX expression - 23 -
3.3.1 Induction test of DsbC-rsNTX in E. coli BL21(DE3) - 23 -
3.4 Protein purification of rsNTX with liquid chromatography - 24 -
3.4.1 rsNTX IMAC purification - 24 -
3.4.2 HPLC profile of rsNTX - 25 -
3.5 rsNTX NMR experiments - 26 -
3.5.1 2D HSQC of rsNTX each peak from HPLC result - 26 -
3.5.2 3D spectra of rsNTX backbone NMR experiments - 26 -
3.6 Recombinant CTXA3 expression - 27 -
3.6.1 Induction test of DsbC-rCTXA3 in E. coli BL21(DE3) - 27 -
3.7 Protein purification of rCTXA3 with liquid chromatography - 28 -
3.7.1 rCTXA3 IMAC purification - 28 -
3.7.2 HPLC profile of rCTXA3 - 29 -
3.8 rCTXA3 NMR experiments - 29 -
3.8.1 2D HSQC of rCTXA3 from HPLC result - 29 -
4 Discussion - 55 -
5 References - 58 -

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