基于溶菌酶的大肠杆菌细胞裂解液优化制备用于无细胞蛋白合成

Lysozyme-Based Escherichia coli Cell Lysate:Optimized Preparation and Application in Cell-Free Protein Synthesis

摘要

无细胞蛋白合成(CFPS)系统因其操作便捷、快速合成蛋白和易于调控的优势,已成为合成生物学研究中的有力平台工具．然而,常规超声裂解法制备细胞裂解液的过程存在设备依赖性强、操作条件不可控、不利于工业放大、易造成裂解液机械或热失活等缺点．基于此,本文提出了一种简单、温和的利用溶菌酶裂解细胞制备大肠杆菌BL21(DE3)裂解液的方法,并对制备过程中各关键条件和工艺进行了优化．即细胞培养过程中采用0.5 mmol/L IPTG诱导产生T7 RNA聚合酶,溶菌酶裂解反应条件为37.0 ℃、pH 7.5、溶菌酶终浓度为1.25 mg/mL；细胞裂解后采用低速离心与振荡孵育相结合收集最终裂解液．蛋白质组学分析表明,这一裂解液制备方法通过保留γ-谷氨酰腐胺氧化还原酶(GGT)及核糖体相关蛋白,有效维持了裂解液的氧化还原稳态及核糖体的翻译活性,为后续CFPS反应提供了稳定的蛋白合成环境．CFPS反应合成增强型绿色荧光蛋白(sfGFP)的结果表明,在较广的DNA模板加入量(50～300 ng)下,采用溶菌酶法制备的细胞裂解液能够达到与常规超声法相当的目标蛋白表达量．该策略为基于裂解液的CFPS系统的简便、可控、低成本构建提供了新方法,有望应用于抗体、酶及其他功能性蛋白产品的体外合成和筛选．

Abstract

Cell-free protein synthesis(CFPS)systems are powerful platforms in synthetic biology,offering operational simplicity,rapid protein production,and tunable reaction conditions. However,the conventional sonication-based cell lysate method is limited by its reliance on specialized equipment,uncontrollable operational parameters,poor scalability,and potential mechanical or thermal inactivation of lysate components. To address these challenges,we developed a mild and streamlined protocol for preparing Escherichia coli BL21(DE3)lysates using lysozyme-based lysis. The following key conditions and processes were systematically optimized:expression of T7 RNA polymerase was induced with 0.5 mmol/L IPTG during cell culture；lysate was conducted at 37.0 ℃ and pH 7.5 using 1.25 mg/mL(final concentration) lysozyme；after cell lysis,the final lysate was collected using a combination of low-speed centrifugation and oscillatory incubation. Proteomic analysis indicated that by preserving γ-glutamylputrescine oxidoreductase(GGT) and ribosome-associated proteins,this preparation method effectively maintained the redox homeostasis and ribosomal translational activity,which provided a stable protein synthesis environment for subsequent CFPS reactions. The results of CFPS reactions producing superfolder green fluorescent protein(sfGFP) showed that lysozyme-based lysates achieved target protein expression levels comparable to conventional sonication-based lysates across a wide range of DNA template concentrations(50—300 ng). This strategy provides a facile,controllable,and cost-effective alternative for constructing CFPS system,with broad potential for in vitro synthesis and screening of antibodies,enzymes,and other functional proteins.

关键词

无细胞蛋白合成 / 大肠杆菌裂解液 / 溶菌酶 / 响应面优化 / 蛋白质组学分析

Key words

cell-free protein synthesis(CFPS) / Escherichia coli lysate / lysozyme / response surface optimization / proteomic analysis

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基金资助

国家自然科学基金资助项目(22178260)

天津市自然科学基金资助项目(24JCYBJC00070)