人源溶菌酶在大肠杆菌中的表达与复性研究

doi:10.13560/j.cnki.biotech.bull.1985.2019-0680

摘要/Abstract

摘要： 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1 170 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1 732 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。

关键词: 人源溶菌酶, 大肠杆菌表达系统, 包涵体, 包涵体体外复性, 溶壁微球菌

Abstract: Human lysozyme（HLZ）is a glycosidolytic enzyme with antibacterial and anti-inflammatory effects. As an alternative to antibiotics,HLZ has been widely used in the food industry,animal husbandry and medical fields. How to obtain HLZ with high yield,high activity and purity is an technical issue to be urgently solved. The codons of HLZ gene were optimized to improve its fitness and expression in Escherichia coli,then the optimized gene of HLZ was introduced into expressing plasmid pET21a and expressed in E. coli expressing strain BL21（DE3）. After inclusion body was dissolved by 8 mol/L urea solution,the effects of 3 refolding methods including one-step dialysis,gradient dialysis and gradient dilution and the concentration of glutathione REDOX（GSSG/GSH）,arginine and glycerol on the refolding of HLZ were investigated to confirm the best refolding plan. Results showed that under the induction by 0.5 mmol/L IPTG at temperature of 37℃,HLZ with the molecular weight of about 14.7 kD was successfully expressed in E. coli,and the expression quantity of inclusion body was about 380 mg/L（wet weight）. After refolding the denatured HLZ by one step dialysis,gradient dialysis and gradient dilution,the specific activities of the enzyme were measured as 147 U/mg,335 U/mg and 176 U/mg respectively,indicating that the optimal refolding method was gradient dialysis. The effects of glutathione redox（GSSG/GSH ratio）,arginine and glycerol concentration on the refolding of HLZ were further investigated. The results showed that the highest enzyme specific activities of refolded HLZ was 1 170 U/mg,which was much higher than the 335 U/mg specific activity of HLZ when the 3 contents were not added,but lower than the enzyme specific activity 1 732 U/mg by commercial HLZ. In conclusion,target HLZ was successfully expressed in E. coli,and recombinant HLZ with high activity was successfully obtained by the refolding system of inclusion body.

Key words: human lysozyme, gene clone, expression system of Escherichia coli, inclusion body, renaturation of inclusion body, Micrococcus lysoleikticus

张春晨, 胡双艳, 阮海华. 人源溶菌酶在大肠杆菌中的表达与复性研究[J]. 生物技术通报, 2020, 36(3): 153-161.

ZHANG Chun-chen, HU Shuang-yan, RUAN Hai-hua. Expression and Renaturation of Recombinant Human Lysozyme in Escherichia coli[J]. Biotechnology Bulletin, 2020, 36(3): 153-161.

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