Nickling Jessica H, Baumann Tobias, Schmitt Franz-Josef, Bartholomae Maike, Kuipers Oscar P, Friedrich Thomas, Budisa Nediljko
Department of Biocatalysis, Institute of Chemistry, Technische Universität Berlin.
Department of Biocatalysis, Institute of Chemistry, Technische Universität Berlin;
J Vis Exp. 2018 May 4(135):57551. doi: 10.3791/57551.
Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) → ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell's resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.
自然界有多种通过修饰单个氨基酸构建块的序列来创造新蛋白质功能的可能性。然而，所有变异都是基于20种标准氨基酸（cAAs）。作为一种将额外物理化学性质引入多肽的方法，非标准氨基酸（ncAAs）的掺入在蛋白质工程中越来越常用。由于其相对较短的长度，通过ncAAs对核糖体合成及翻译后修饰的肽进行修饰尤其具有吸引力。通过对单个残基进行特定修饰，可以产生新的功能和化学手柄。选择性压力掺入（SPI）方法利用在化学限定生长培养基中被剥夺必需氨基酸的营养缺陷型宿主菌株。然后，几种结构和化学相似的氨基酸类似物可以被相应的氨酰 - tRNA合成酶激活，并在目标肽或蛋白质序列中提供残基特异性的cAA(s)→ncAA(s)替换。尽管在SPI方法的背景下，ncAAs在重组基因表达阶段也会掺入宿主蛋白质组，但细胞的大部分资源都分配给了目标基因的表达。这使得能够高效地进行残基特异性的ncAAs掺入，通常伴随着大量修饰后的目标产物。本文描述了六种脯氨酸类似物在体内掺入乳酸乳球菌天然产生的羊毛硫抗生素——乳链菌肽中的情况。在营养缺陷型大肠杆菌菌株中于限定生长培养基中进行发酵和表达时，乳链菌肽的抗菌特性可以发生改变并进一步扩展。因此，用ncAAs残基特异性替换cAAs的作用可以带来抗菌活性和特异性的变化。抗菌活性测定和荧光显微镜用于测试新的乳链菌肽变体对革兰氏阳性乳酸乳球菌指示菌株的生长抑制作用。质谱用于确认生物活性乳链菌肽变体中ncAAs的掺入情况。
