重组蓖麻毒素A链的表达及半乳糖修饰

Expression and galactose modification of recombinant ricin toxin A chain

目的表达重组蓖麻毒素A链(ricin toxin A,RTA),并进行半乳糖糖基化修饰。方法将重组质粒pET-28a-RTA转化E. coli BL21(DE3),经IPTG诱导表达重组蛋白RTA,通过镍离子亲和层析柱纯化包涵体,并复性。采用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐[1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,EDC]/N,N-羟基琥珀酰亚胺(N-hydroxysuccinimide,NHS)法对重组RTA进行半乳糖糖基化修饰,并对偶联反应温度(20、25、30和37℃)和半乳糖与RTA的投料比(100∶1、500∶1、1 000∶1、1 500∶1和2 000∶1)进行优化。结果 RTA蛋白相对分子质量约34 000,主要以包涵体形式表达,纯化复性后纯度大于90%。最适偶联反应温度为25℃,半乳糖与RTA最适投料比为1 000∶1。结论成功表达了RTA蛋白,并通过EDC/NHS法实现了半乳糖与RTA的偶联。

Objective To express recombinant ricin toxin A chain(RTA) and modify by glycosylation with galactose.Methods Recombinant plasmid pET-28 a-RTA was transformed to E. coli BL21(DE3)and induced by IPTG. The expressed RTA was purified by nickel ion affinity chromatography and refolded,then modified by galactose glycosylation using 1-(3-dimethylaminoprophyl)-3-ethylcarbodiimide hydrochloride(EDC)/N-hydroxysuccinimide(NHS),and the temperature for coupling reaction(20,25,30 and 37 ℃) and the ratio of galactose to RTA(100 ∶ 1,500 ∶ 1,1 000 ∶ 1,1 500 ∶ 1 and 2 000 ∶ 1)were optimized. Results The expressed RTA with a relative molecular mass of about 34 000 was mainly in a form of inclusion body,of which the purity was more than 90%. The optimal temperature for coupling reaction was25 ℃,while the optimal ratio of galactose to RTA was 1 000 ∶ 1. Conclusion RTA was expressed successfully and coupled to galactose by EDC/NHS method.