氨基酸定点突变提高灵芝蛋白LZ-8热稳定性的研究

通过氨基酸定点突变技术提高灵芝免疫调节蛋白LZ-8的热稳定性。通过分子动力学模拟结合温度因子预测对LZ-8氨基酸突变位点进行理性设计,在毕赤酵母X33菌株内构建并表达LZ-8突变体蛋白,采用HeLa细胞生长抑制实验和差示量热扫描分析检测并比较了LZ-8突变前后生物学活性及热力学参数。结果显示,LZ-8 N-端α螺旋为理论预测的温度敏感区域,在该区域进行F8W和R9K氨基酸双位点突变,突变后的LZ-8热稳定性提高,相变温度Tm上升0.92℃,相转变焓值ΔH提高23.14 kJ/mol,但突变后LZ-8生物学活性基本不变,LZ-8和LZ-8突变体对HeLa细胞生长抑制的IC50值分别是2.238μg/mL和2.407μg/mL。通过理性设计氨基酸突变位点,获得了稳定性提高但生物学活性不变的灵芝免疫调节蛋白LZ-8的突变体。

Improvement of Thermal Stability of Ganoderma lucidum Protein LZ-8 by Site-directed Mutation of Amino Acids

This work is to improve the thermal stability of Ganoderma lucidum immunoregulatory protein LZ-8 by site-directed mutagenesis of amino acids.Molecular dynamics simulation combined with temperature factor prediction were used to reasonably design LZ-8 amino acid mutation site,LZ-8 mutant protein was constructed and expressed in Pichia pastoris X33 strain,the biological activity and thermodynamic parameters of LZ-8 before and after mutation were detected and compared by HeLa cell growth inhibition experiment and differential scanning calorimetric(DSC)study.Results showed that LZ-8 N-terminal alpha helix was the temperature sensitive region predicted by theory.F8W and R9K double site mutations occurred in this region.After mutation,the thermal stability of LZ-8 was improved,the phase transition temperature Tm increased by 0.92℃,and the phase transition enthalpyΔH increased by 23.14 kJ/mol.However,the biological activity of LZ-8 after mutation was basically unchanged.The IC 50 of LZ-8 and LZ-8 mutant on HeLa cell growth inhibition was 2.238μg/mL and 2.407μg/mL,respectively.Ganoderma lucidum LZ-8 mutant with improved stability but unchanged biological activity is obtained by rational design of amino acid mutation sites.