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首页> 外国专利> METHOD OF WHEAT CYSTEINE PROTEASE PROTEINS FAMILY (TRITICUM AESTIVUM) PRODUCING AND PREPARATION OF TRITIKAIE-ALPHA PROTEIN, OBTAINED USING SAID METHOD

METHOD OF WHEAT CYSTEINE PROTEASE PROTEINS FAMILY (TRITICUM AESTIVUM) PRODUCING AND PREPARATION OF TRITIKAIE-ALPHA PROTEIN, OBTAINED USING SAID METHOD

机译：小麦半胱氨酸蛋白酶家族蛋白的制备和制备方法的研究

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FIELD: biotechnology.;SUBSTANCE: invention relates to biotechnology and represents method of tritikaine-alpha wheat truncated shape producing, having sequence of SEQ ID NO: 2 (shortTRIT-α), recombinant expressed in bacterial system, consisting in culturing of cells E.coli JM109, transformed by plasmid pQE80L_shortTRIT-α, containing DNA sequence coding shortTRIT-α protein, in LB medium with addition of ampicillin at 37 °C under aerobic conditions for 12-14 h, nutrient medium is inoculated with inoculum, culture is raised until optical density of A600 0.6-0.8, induced by 1 mM isopropylite-β-D-galactoside and raised for another 2.5-3 hours; target protein shortTRIT-α cleaning is carried out by affine metal-chelate chromatography: deposited by centrifugation expression culture cell biomass is re-suspended in buffer containing 0.01 M of Tris-HCl, pH 8.0 and homogenized on ultrasonic disintegrator for 1 min at 4 °C, produced after lysate centrifugation residue is washed with initial buffer and dissolved in buffer A, consisting of 6 M of guanidine-chloride and 0.05 M of Tris-HCl, pH 7.8, solution is clarified by centrifugation and applied on column with activated nickel ions iminodiacetate-sepharose, balanced by buffer A, sorbent is successively washed with balancing buffer A and same buffer containing 8 M of urea and 0.005 M of imidazole, protein is eluted with buffer A containing 8 M of urea and 0.25 M of imidazole, then eluate is added into cooled buffer of 0.05 M of Tris-HCl, 0.5 M of arginine-chloride, 2 M of urea, pH 7.8 at ratio of 1:5 and stirred for 1 h at 4 °C, solution is dialyzed against 0.05 M of Tris-HCl, pH 7.8 at 4 °C, supernatant produced after dialysate centrifugation, is concentrated on Amicon cell with RM-10 (Millipore) membrane, with subsequent dialysis against phosphate-salt buffer PBS, pH 7.4, at 4 °C, concentration of shortTRIT-α protein is determined, aliquoted by glass bottles, freezing and lyophilized.;EFFECT: invention enables to obtain pure protein preparation with tritikaine-alpha wheat activity with high degree of efficiency.;3 cl, 6 dwg, 4 ex
机译：具有SEQ ID NO：2（shortTRIT-α）序列的曲替卡因-α小麦截短形状的生产方法，涉及在细菌系统中表达的重组生物技术，涉及培养E细胞。大肠杆菌JM109，经质粒pQE80L_shortTRIT-α转化，含有编码shortTRIT-α蛋白的DNA序列，在LB培养基中于37°C在有氧条件下加入氨苄西林，在有氧条件下培养12-14 h，将营养培养基接种接种物，培养直至1 mM异丙基-β-D-半乳糖苷诱导的A 600 的光密度为0.6-0.8，再升高2.5-3小时;靶蛋白shortTRIT-α的清洁通过仿射金属螯合层析进行：通过离心表达沉积，将细胞生物质重悬于含有0.01 M Tris-HCl，pH 8.0的缓冲液中，并在超声分解仪上于4°C均质1分钟裂解物离心分离后产生的C溶液用初始缓冲液洗涤并溶解于缓冲液A中，该缓冲液A由6 M的氯化胍和0.05 M的Tris-HCl（pH 7.8）组成，通过离心分离将溶液澄清，并用活化的镍离子应用于色谱柱亚氨基二乙酸-琼脂糖，用缓冲液A平衡，吸附剂依次用平衡缓冲液A和含8 M尿素和0.005 M咪唑的相同缓冲液洗涤，蛋白用含8 M尿素和0.25 M咪唑的缓冲液A洗脱，然后洗脱将其以1：5的比例加入到冷却的缓冲液中，该缓冲液中含有0.05 M的Tris-HCl，0.5 M的精氨酸-氯化物，2 M的尿素，pH 7.8，并在4°C搅拌1 h，将溶液与0.05 M pH 7.8的Tris-HCl在4°C下，透析液离心后产生的上清液在带有RM-10（Millipore）膜的Amicon细胞上浓缩，随后在4°C下用磷酸盐溶液PBS，pH 7.4透析，shortTRIT-α蛋白的浓度为测定;通过玻璃瓶等分，冷冻并冻干。;效果：本发明能够以高效率获得具有曲替卡因-α小麦活性的纯蛋白制剂。3cl，6 dwg，4 ex

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