Highly efficient soluble expression and purification of recombinant human basic fibroblast growth factor (hbFGF) by fusion with a new collagen-like protein (Scl2) in Escherichia coli

Abstract

Human basic fibroblast growth factor (hbFGF) is involved in a wide range of biological activities that affect the growth, differentiation, and migration. Due to its wound healing effects and therapy, hbFGF has the potential as therapeutic agent. Therefore, large-scale production of biologically active recombinant hbFGF with low cost is highly desirable. However, the complex structure of hbFGF hinders its high-level expression as the soluble and functional form. In the present study, an efficient, cost-effective, and scalable method for producing recombinant hbFGF was developed. The modified collagen-like protein (Scl2-M) from Streptococcus pyogenes was used as the fusion tag for producing recombinant hbFGF for the first time. After optimization, the expression level of Scl2-M-hbFGF reached approximately 0.85?g/L in the shake flask and 7.7?g/L in a high cell-density fermenter using glycerol as a carbon source. Then, the recombinant Scl2-M-hbFGF was readily purified using one-step acid precipitation and the purified Scl2-M-hbFGF was digested with enterokinase. The digested mixture was further subject to ion-exchange chromatography, and the final high-purity (96%) hbFGF product was prepared by freeze-drying. The recovery rate of the whole purification process attained 55.0%. In addition, the biological activity of recombinant hbFGF was confirmed by using L929 and BALB/c3T3 fibroblasts. Overall, this method has the potential for large scale production of recombinant hbFGF.     

人源溶菌酶在大肠杆菌中的表达与复性研究

人源溶菌酶(Human lysozyme,HLZ)是一种糖苷水解酶,具有抗菌消炎的作用,其作为抗生素的替代品,已经被广泛应用于食品业、畜牧业和医疗等领域。如何获得高产量、高活性、高纯度的人源溶菌酶一直是亟待解决的技术问题。优化人源溶菌酶编码基因密码子,提高其在大肠杆菌中的适应度和表达量;将优化的基因克隆至大肠杆菌表达质粒pET21a,并将其在大肠杆菌表达菌株BL21(DE3)中诱导表达;利用8 mol/L尿素溶液对包涵体进行溶解变性后,探究一步透析、梯度透析和梯度稀释3种复性方式以及复性液中谷胱甘肽氧化还原对(GSSG/GSH)、精氨酸、甘油等复性物的浓度对重组人源溶菌酶复性的效果,获得最佳的复性方案。研究结果表明:37℃诱导温度下,利用0.5 mmol/L IPTG成功诱导了分子量约为14.7 kD的重组人源溶菌酶的表达,包涵体表达量约为380 mg/L(湿重)。包涵体经一步透析、梯度透析和梯度稀释3种复性方式复性后,测得比活力值分别为147 U/mg、335 U/mg、176 U/mg,表明最佳复性方法为梯度透析复性法。进一步探索了复性液中GSSG/GSH比值、精氨酸浓度、甘油浓度对人源溶菌酶复性效果的影响,表明当复性液中同时添加浓度比为1∶2的GSSG/GSH、4 mmol/L精氨酸和6%甘油时,复性后人源溶菌酶的最佳比活力值为1170 U/mg,显著高于3种复性物均不加时溶菌酶335 U/mg的比活力值,但低于溶菌酶标准品1732 U/mg的比活力值。成功地将人源溶菌酶基因在大肠杆菌中表达,并通过包涵体复性体系成功获得高活性重组人源溶菌酶。     

阑尾切除术后切口感染患儿PCT、ESR和 CRP变化及切口脓液病原菌分析

目的研究阑尾切除术后切口感染患儿血清C-反应蛋白(CRP)、降钙素原(PCT)和红细胞沉降率(ESR)水平变化及切口脓液病原菌分布。方法选取2014年7月至2019年7月我院收治的100例阑尾切除术后切口感染患儿为观察组,对照组选取同期来我院进行体检的80例健康儿童。对比两组对象血清CRP、PCT和ESR水平,同时分析观察组患儿阑尾切除术后切口脓液病原菌的构成。结果观察组患儿血清CRP、PCT和ESR水平显著高于对照组(均P0.05)。送检的100例脓液及脓性分泌物标本中检出细菌51例,检出率为51.00%。共分离得到病原菌55株(其中有4例为2种细菌混合感染),病原菌种类共13种,其中革兰阴性菌39株(70.91%),革兰阳性菌16株(29.09%),主要病原菌为大肠埃希菌(49.09%)、肺炎克雷伯菌(10.91%)、金黄色葡萄球菌(9.09%)和铜绿假单胞菌(5.45%)。大肠埃希菌、肺炎克雷伯菌和铜绿假单胞菌对厄他培南、亚胺培南、美罗培南、哌拉西林/他唑巴坦、氨曲南较为敏感。结论阑尾切除术后切口感染患儿血清PCT、CRP和ESR水平较高,主要病原菌为大肠埃希菌。     

儿童反复呼吸道感染与肠道微生态变化的相关性

目的研究儿童反复呼吸道感染与患儿肠道微生态平衡紊乱的关系。方法选择102例反复呼吸道感染患儿为研究组,167例急性肺炎患儿为肺炎对照组,142例健康体检儿童为正常对照组。采用16S rRNA荧光定量PCR检测3组对象肠道双歧杆菌及大肠埃希菌数量,计算B/E值,并比较3组研究对象细胞免疫功能。结果正常对照组、肺炎对照组以及研究组儿童肠道双歧杆菌数量依次降低,大肠埃希菌依次增多,B/E值依次降低,差异均有统计学意义(均P0.05)。正常对照组、肺炎对照组以及研究组儿童血液中CD3~+、CD4~+细胞水平以及CD4~+/CD8~+依次降低,CD8~+细胞水平依次增高,差异均有统计学意义(均P0.05)。结论儿童反复呼吸道感染与肠道微生态失衡具有一定相关性,肠道微生态稳态的维持可为反复呼吸道感染的防治提供新思路。     

提高大肠杆菌表达外源蛋白胞外含量的策略

大肠杆菌的蛋白质表达平台在工业和农业中得到了广泛应用,使目的蛋白质表达后释放至胞外更有利于大规模的生产。目前,已经研究出许多改善外源蛋白胞外含量的方法。本文从蛋白质分泌机制、菌株、信号肽、载体和培养条件的选择和优化改造、密码子的优化和蛋白质跨膜转运过程的改善等方面总结了提高大肠杆菌表达外源蛋白的胞外含量的各种策略,指出多因素协同作用才能更全面地提升蛋白质的胞外产量。     

Variation in the ratio of curli and phosphoethanolamine cellulose associated with biofilm architecture and properties

Bacterial biofilms are communities of bacteria entangled in a self‐produced extracellular matrix (ECM). Escherichia coli direct the assembly of two insoluble biopolymers, curli amyloid fibers, and phosphoethanolamine (pEtN) cellulose, to build remarkable biofilm architectures. Intense curiosity surrounds how bacteria harness these amyloid‐polysaccharide composites to build biofilms, and how these biopolymers function to benefit bacterial communities. Defining ECM composition involving insoluble polymeric assemblies poses unique challenges to analysis and, thus, to comparing strains with quantitative ECM molecular correlates. In this work, we present results from a sum‐of‐the‐parts ¹³C solid‐state nuclear magnetic resonance (NMR) analysis to define the curli‐to‐pEtN cellulose ratio in the isolated ECM of the E. coli laboratory K12 strain, AR3110. We compare and contrast the compositional analysis and comprehensive biofilm phenotypes for AR3110 and a well‐studied clinical isolate, UTI89. The ECM isolated from AR3110 contains approximately twice the amount of pEtN cellulose relative to curli content as UTI89, revealing plasticity in matrix assembly principles among strains. The two parent strains and a panel of relevant gene mutants were investigated in three biofilm models, examining: (a) macrocolonies on agar, (b) pellicles at the liquid‐air interface, and (c) biomass accumulation on plastic. We describe the influence of curli, cellulose, and the pEtN modification on biofilm phenotypes with power in the direct comparison of these strains. The results suggest that curli more strongly influence adhesion, while pEtN cellulose drives cohesion. Their individual and combined influence depends on both the biofilm modality (agar, pellicle, or plastic‐associated) and the strain itself.