Department of Applied Chemistry, Yamaguchi University, Tokiwadai 2-16-1, Ube 755-8611, Japan.
ACS Appl Bio Mater. 2024 Aug 19;7(8):5566-5578. doi: 10.1021/acsabm.4c00670. Epub 2024 Jul 15.
Papain is useful for the enzymatic digestion of various proteins to produce functional peptides or protein fragments. Immobilized papain being reactive toward proteins and easily removable from a reaction mixture is worth developed. In the present work, liposomes were applied as colloidal carriers of papain for the catalytic digestion of polyclonal immunoglobulin G (IgG). Papain was covalently conjugated at pH = 7.0 via tris-succinimidyl aminotriacetate (TSAT) to liposomes incorporated with 5 mol % poly(ethylene glycol)-tethered lipid with a reactive amino group. The papain-conjugated liposome (liposome-papain) catalyzed the hydrolysis of -benzoyl-l-arginine 4-nitroanilide hydrochloride (BAPNA) at pH = 5.0-7.0. The activity of liposome-papain significantly increased with increasing temperature from 25 to 50 °C. The Michaelis constant was determined with respect to the liposome-papain- and free papain-catalyzed reactions with BAPNA at 37 °C as = 1.11 ± 0.13 and 11.6 ± 2.9 mM, respectively. Liposome-papain was applied to the catalytic digestion of 10 mg·mL IgG at 37 °C for 24 h at pH = 5.0-7.0. The reaction mixture could be analyzed without pretreatment by using the affinity columns immobilized with the protein A or protein L ligand because colloidal liposome-papain quickly flowed through the chromatographic stationary phase, exhibiting little proteolytic effect on the proteinaceous ligands. The analysis clearly demonstrated the catalytic production of antigen-binding fragments (Fab) from IgG in an enzyme concentration- and pH-dependent manner. Liposome-papain with 15 or 50 mol % anionic lipids also catalyzed the formation of Fab from IgG. The above results demonstrated that liposome-papain was useful to digest IgG and to purify Fab formed with the affinity chromatography.
木瓜蛋白酶可用于各种蛋白质的酶解，以产生功能性肽或蛋白质片段。固定化木瓜蛋白酶对蛋白质具有反应性，且易于从反应混合物中去除，因此值得开发。本工作将脂质体用作木瓜蛋白酶的胶体载体，用于催化多克隆免疫球蛋白 G (IgG)的酶解。通过三(琥珀酰亚胺基)氨基三乙酸酯（TSAT）在 pH = 7.0 下将木瓜蛋白酶共价偶联到与 5 mol %聚乙二醇-连接脂质结合的脂质体上，该脂质带有反应性氨基。在 pH = 5.0-7.0 下，偶联到脂质体上的木瓜蛋白酶（脂质体-木瓜蛋白酶）催化 -苯甲酰-L-精氨酸 4-硝基苯胺盐酸盐（BAPNA）的水解。脂质体-木瓜蛋白酶的活性随温度从 25°C 增加到 50°C而显著增加。在 37°C 下，通过 BAPNA 测定的脂质体-木瓜蛋白酶和游离木瓜蛋白酶催化反应的米氏常数分别为 = 1.11 ± 0.13 和 11.6 ± 2.9 mM。在 pH = 5.0-7.0 下，将脂质体-木瓜蛋白酶应用于 10 mg·mL IgG 的催化消化，在 37°C 下反应 24 h。反应混合物无需预处理即可通过固定化蛋白质 A 或蛋白质 L 配体的亲和柱进行分析，因为胶体脂质体-木瓜蛋白酶快速流过色谱固定相，对蛋白质配体几乎没有蛋白水解作用。分析清楚地表明，在酶浓度和 pH 依赖性方式下，从 IgG 中催化产生抗原结合片段（Fab）。带 15 或 50 mol %阴离子脂质的脂质体-木瓜蛋白酶也可催化 IgG 形成 Fab。上述结果表明，脂质体-木瓜蛋白酶可用于消化 IgG，并通过亲和色谱法纯化形成的 Fab。
