热处理对海参溶菌酶C端多肽的抑菌活性和结构的影响

doi:10.7503/cjcu20131144

摘要/Abstract

摘要：

实验优化了重组蛋白C端多肽(SjLys-C)的E. coli Rosetta(DE3)pLysS表达系统, 将重组质粒pET-32a(+)-SjLys-C转入有助于蛋白二硫键正确折叠的新表达宿主E. Coli Rosetta-GamiB(DE3)pLysS中. SDS-PAGE分析表明, 重组蛋白SjLys-C在宿主中得到高效可溶表达. 抑菌实验结果表明, 重组蛋白SjLys-C具有较高的抑菌活性; 经100 ℃处理40 min后, 蛋白SjLys-C的抑菌活性提高. 分子动力学(MD)模拟表明, SjLys-C蛋白具有高度的热稳定性, 蛋白热处理后未变性, 仍维持完整的三级结构. 但在热处理过程中, SjLys-C多肽的氨基酸残基随温度变化发生内部结构的重排. 其中C端区、 N端区和活性区域(10~20)内氨基酸残基的柔性增加, 蛋白整体构象展开, 导致被包埋在内部的2个活性位点(Ser18, His48)暴露, 并且它们之间的距离缩短; 而活性区域(37~47)内氨基酸残基的构象调整, 导致区域结构紧凑, 使2个活性位点间的距离改变.

关键词: 海参溶菌酶, 重组蛋白SjLys-C, 抑菌活性, 热处理, 分子动力学模拟

Abstract:

The recombinant polypeptide derived from C-terminal domain of the lysozyme from the sea cucumber Stichopus japonicus(SjLys-C) was used to detect the effect of temperature variation on the antibacterial activity and its structural change. The E. coli Rosetta-gamiB(DE3) pLysS was chosen as the new expression host to gain the recombinant C-terminal polypeptide of sea cucumber lysozyme successfully. Based on the antibacterial test, the fusion protein SjLys-C produced by the new host displayed potent inhibitive effect on the growth of M. lysodeikticus, S. aureus, P. aeruginosa and V. parahaemolyticus. In addition, the inhibitive activities about three tested bacteria were enhanced about 5%—22% after the fusion protein heated in 100 ℃ for 40 min. The molecule dynamic(MD) simulation was made to discover the conformational changes of SjLys-C after the heated treatment. The molecule dynamic simulation demonstrated that SjLys-C remained a stable conformation under the higher temperature condition. But several parts of amino acid residues on the SjLys-C were rearranged and the space distance between two active sites of Ser18 and His48 was closer at 100 ℃. The results might discover a new interpretation of the antibacterial function under the heated treatment of the sea cucumber lysozyme polypeptide.

Key words: Lysozyme of sea cucumber, Recombinant protein SjLys-C, Antibacterial activity, Heated treatment, Molecule dynamic simulation

中图分类号:

O641
Q786

TrendMD:

武瑶, 梁雯婧, 李成, 商旭, 丛丽娜. 热处理对海参溶菌酶C端多肽的抑菌活性和结构的影响. 高等学校化学学报, 2014, 35(5): 1044.

WU Yao, LIANG Wenjing, LI Cheng, SHANG Xu, CONG Lina. Effects of Temperature on the Antibacterial Activity and Structural Change of the C-terminal Polypeptide of the Sea Cucumber Lysozyme^†. Chem. J. Chinese Universities, 2014, 35(5): 1044.

图/表 7

Fig.1 Map of the recombination plasmid(A) and the expression of recombination SjLys-C on SDS-PAGE(B)^M: Protein markers; lane 1: control; lane 2: total cellular protein; lane 3: sample in precipitation after ultrasonication; lane 4: sample in supernatant after ultrasonication; lane 5: recombinant SjLys-C after Ni2+-NTA affinity chromatography; lane 6: recombinant SjLys-C after G-25 desalination column.

Table 1 Antimicrobial activities of the recombinant SjLys-C expressed in different host strains

Tested bacteria	Diameter of inhibition zone^a/mm
	Rosetta(DE3)pLysS^b		Rosetta-gamiB(DE3)pLysS
	SjLys-C	Heat-treated SjLys-C	SjLys-C	Heat-treated SjLys-C
M. lysodeikticus	19	23	18	22
S. aureus	10	10	19	19
P. aeruginosa	8	8	14	15
V. parahaemolyticus	20	21	20	21

Fig.2 Antimicrobial activities of recombinant protein SjLys-C(A) and the samples after the heat treatment(B)^(A) M. lysodeiktieus; (B) P. aeruginosa.

Fig.3 Time evolution of RMSD(A) and Rg(B) of MD structure of SjLys-C at 30 ℃(a) and 100 ℃(b), respectively

Fig.4 Topological graphs for the secondary structures(A) and the MD images of conformational structure(B) for SjLys-C at 30 and 100 ℃

Fig.5 Time evolution of RMSF of MD structure of SjLys-C at 30 ℃(a) and 100 ℃(b), respectively

Fig.6 Comparision of the structures from simulated annealing^The native sructures were selected from the MD structure of SjLys-C at 100 ℃ at different time points 5, 6, 7, 8, 9 and 10 ns.

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