N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098 Moscow, Russia.
Lomonosov Moscow State University, 119991 Moscow, Russia.
Biomolecules. 2020 Mar 12;10(3):440. doi: 10.3390/biom10030440.
The use of recombinant endolysins is a promising approach for antimicrobial therapy capable of counteracting the spread of antibiotic-resistant strains. To obtain the necessary biotechnological product, diverse peptide tags are often fused to the endolysin sequence to simplify enzyme purification, improve its ability to permeabilize the bacterial outer membrane, etc. We compared the effects of two different types of protein modifications on endolysin LysECD7 bactericidal activity in vitro and demonstrated that it is significantly modulated by specific permeabilizing antimicrobial peptides, as well as by widely used histidine tags. Thus, the tags selected for the study of endolysins and during the development of biotechnological preparations should be used with the appropriate precautions to minimize false conclusions about endolysin properties. Further, modifications of LysECD7 allowed us to obtain a lytic enzyme that was largely devoid of the disadvantages of the native protein and was active over the spectra of conditions, with high in vitro bactericidal activity not only against Gram-negative, but also against Gram-positive, bacteria. This opens up the possibility of developing effective antimicrobials based on N-terminus sheep myeloid peptide of 29 amino acids (SMAP)-modified LysECD7 that can be highly active not only during topical treatment but also for systemic applications in the bloodstream and tissues.
使用重组内切溶素是一种有前途的抗菌治疗方法，能够对抗抗生素耐药菌株的传播。为了获得必要的生物技术产品，通常将各种肽标签融合到内切溶素序列中，以简化酶的纯化，提高其穿透细菌外膜的能力等。我们比较了两种不同类型的蛋白质修饰对内切溶素 LysECD7 体外杀菌活性的影响，结果表明，它可以被特定的渗透抗菌肽以及广泛使用的组氨酸标签显著调节。因此，在研究内切溶素和开发生物技术制剂时选择的标签应谨慎使用，以尽量减少对内切溶素特性的错误结论。此外，LysECD7 的修饰使我们获得了一种溶酶酶，该酶在很大程度上没有天然蛋白的缺点，并且在各种条件下都具有活性，体外杀菌活性高，不仅对革兰氏阴性菌有效，而且对革兰氏阳性菌也有效。这为开发基于 N 端 29 个氨基酸的绵羊髓鞘碱性蛋白（SMAP）修饰的 LysECD7 的有效抗菌剂开辟了可能性，这种抗菌剂不仅在局部治疗时具有高度活性，而且在血流和组织中的全身应用时也具有高度活性。
