重组溶葡萄球菌酶的PEG定点修饰

Site-specific PEGylation of recombinant lysostaphin

摘要:

为获得高抗菌活性聚乙二醇 (PEG) 定点修饰的溶葡萄球菌酶 (Lysn)，根据该酶的高级结构，在它的催化域和结合域上优选8个位点 (Q9、N13、N40、T172、N174、G197、V240和T244)，将其分别突变成半胱氨酸，纯化后的溶葡萄球菌酶突变体经DTT处理后与20 kDa的单甲氧基聚乙二醇马来酰亚胺 (mPEG-MAL)进行定点修饰反应，RP-HPLC分析显示PEG修饰率大于70%，修饰产物经MacroCap SP阳离子交换层析纯化后，PEG化溶葡萄球菌酶的纯度大于95%。比浊法和最小抑菌浓度 (MIC) 实验表明20K-PEG-LysnV240C和20K-PEG-LysnT244C的抗菌活性维持在原来的50%左右。研究结果为溶葡萄球菌酶全身给药治疗金黄色葡萄球菌感染奠定基础。

Abstract:

Lysostaphin (Lysn) is an antibacterial metalloendopeptidase that cleaves the pentaglycin bridges in the cell wall of Staphylococci. Although many studies have demonstrated its high activity in vitro, the medical application of Lysn has been hampered by its short half-life in vivo. In order to enhance its stability in vivo without significantly suppressing the enzymatic activity, we designed and tested eight single cysteine substitutions in Lysn for covalent attachment of polyethylene glycol chains (PEGylation). The purified mutants, fully reduced by Dithiothreitol (DTT), were treated with mPEG-MAL(20 kDa). The PEG modification efficiency was above 70% as determined by reverse-phase high-pressure liquid chromatography (HPLC) analysis. The PEG-Lysn proteins were further purified by cation exchange chromatography (MacroCap SP), reaching at least 95% purity. The activities of the PEG-Lysn proteins were determined by the turbidity and minimum inhibitory concentration (MIC) assays. We found that the PEGylated V240C and T244C mutants retained about 50% of the original antibacterial activity of Lysn. Overall, this study will help develop highly stable and active PEG-Lysn to treat systemic S. aureus infections.