重組鱟血漿凝集素2之細菌及配體結合活性探討

Characterization of bacteria and ligand binding activities of recombinant Tachypleus plasma lectin 2

劉俞伶(Low, Ee Ling)

指導教授 ： 張大慈

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2010年)

摘要

由台灣種鱟 (Taiwanese Tachypleus tridentatus) 的血漿中分離出之血漿凝集素 2 (TPL-2) 具有辨識格蘭氏陽性及陰性菌的功能。以往利用嗜甲醇酵母菌 (Pichia pastoris) 表現TPL-2之表現量及產率皆低，因此，結合親和性融合蛋白標誌以提升 TPL-2 的表現量，並探討其生物功能為本論文主要目標。本研究利用蛋白質工程技術設計TPL-2分子，使之具備胺基端的米根黴菌澱粉吸附區域 (Rhizopus oryzae starch-binding domain, RoSBD)、米根黴菌葡萄糖水解酵素連接片段 (Rhizopus oryzae glucoamylase linker, RoLK)、以及羧基端的台灣種類鱟血漿凝集素 2 組成ALT2，繼而將其蛋白質序列中兩個N-醣基化位置修飾，使天門冬氨酸 (Asparagine, Asn) 140 及 150皆突變成天門冬氨酸鹽 (Asparatate, Asp)之ALT2-dm。運用此新穎的重組嗜甲醇酵母菌進行工業等級大量表現ALT2-dm，能以吸附澱粉的方式純化，且純化後之ALT2-dm產量可達到每公升79.8 毫克。其生物功能性分析顯示ALT2-dm 能識別特定的細菌並結合病原相關分子型態 (pathogen associated molecular patterns, PAMPs)，例如格蘭氏陽性菌的脂胞壁酸 (lipoteichoic acid, LTA) 及格蘭氏陰性菌的脂多醣 (lipopolysaccharide, LPS) 。此外，亦發現 ALT2-dm 能辨識原核生物細胞壁中保留性單醣分子結構，繼而發展高靈敏性之檢測方法。本論文之具體貢獻為大量表現鱟血漿凝集素以進行功能分析、修飾蛋白質的醣基化官能基以探討蛋白質與醣配體結合個性、及發現重組鱟血漿凝集素之新穎功能。

關鍵字

米根黴菌澱粉吸附區域 ； 鱟血漿凝集素 2 ； 脂多醣

並列摘要

Tachypleus plasma lectin two (TPL-2) derived from the hemolymph of Taiwanese horseshoe crab, has been found to be able to recognize Gram-positive and negative bacteria. Previously, recombinant TPL-2 was expressed by yeast P. pastoris, but the expression level and purification yield were low. Therefore, the aim of this study is to increase TPL-2 expression by fusing an affinity tag for investigation of biological functions. Recombinant TPL-2 was designed and engineered to be consisted of an N-terminal Rhizopus oryzae starch binding domain (RoSBD), an R. oryzae glucoamylase linker (RoLK), and a C-terminal TPL-2 to give ALT2. ALT2 was further modified on two N-glycosylation sites at Asn 140 and 150 to make ALT2-dm. ALT2-dm was overexpressed in P. pastoris from laboratory scale up to 100 L, and starch matrix adsorption was proven to be feasible for the purification of and a purification yield of 79.8 mg/L was achieved. Functional analyses revealed that ALT2-dm recognized specific bacteria and the pathogen associated molecular patterns (PAMPs) such as lipoteichoic acid (LTA) in Gram-positive bacteria and lipopolysaccharide (LPS) in Gram-negative bacteria. In addition, ALT2-dm was capable of recognizing a conserved moiety in the prokaryotic cell wall which in turn led to the development of high sensitivity detection methods. The major contribution of this study include achievement of high level expression of TPL-2, modulation of protein glycosylation, characterization of protein-glycan interaction, and discovery of a novel function of recombinant TPL-2.

並列關鍵字

Rhizopus oryzae starch binding domain ； Tachypleus plasma lectin 2 ； lipopolysaccharide

參考文獻

Adler, P., Wood, S.J., Lee, Y.C., Lee, R.T., Petri, W.A., Jr. and Schnaar, R.L. (1995) High affinity binding of the Entamoeba histolytica lectin to polyvalent N-acetylgalactosaminides. J Biol Chem 270, 5164-5171.

Andres, E., Molinari, J., Peterszegi, G., Mariko, B., Ruszova, E., Velebny, V., Faury, G. and Robert, L. (2006) Pharmacological properties of rhamnose-rich polysaccharides, potential interest in age-dependent alterations of connectives tissues. Pathol Biol (Paris) 54, 420-425.

Arnau, J., Lauritzen, C., Petersen, G.E. and Pedersen, J. (2006) Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr Purif 48, 1-13.

Arsenault, T.L., Maclean, D.B., Zou, W. and Szarek, W.A. (1991) Structural studies on the polysaccharide portion of "A-band" lipopolysaccharide from a mutant (AK1401) of Pseudomonas aeruginosa strain. Canadian Journal of Chemistry 69, 1273-1280.

Bundle, D.R., Baumann, H., Brisson, J.R., Gagne, S.M., Zdanov, A. and Cygler, M. (1994) Solution structure of a trisaccharide-antibody complex: comparison of NMR measurements with a crystal structure. Biochemistry 33, 5183-5192.