Plavec Tina Vida, Štrukelj Borut, Berlec Aleš
Department of Biotechnology, Jožef Stefan Institute, Ljubljana, Slovenia.
Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia.
Front Microbiol. 2019 Aug 13;10:1879. doi: 10.3389/fmicb.2019.01879. eCollection 2019.
The display of recombinant proteins on bacterial surfaces is a developing research area with a wide range of potential biotechnological applications. The lactic acid bacterium is an attractive host for such surface display, and a promising vector for delivery of bioactive proteins. Surface-displayed recombinant proteins are usually anchored to the bacterial cell wall through anchoring domains. Here, we investigated alternatives to the commonly applied lactococcal lysine motif (LysM)-containing surface anchoring domain, the C-terminus of AcmA (cAcmA). We screened 15 anchoring domains of lactococcal or phage origins that belong to the Pfam categories LPXTG, LysM, CW_1, Cpl-7, WxL, SH3, and ChW, which can provide non-covalent or covalent binding to the cell wall. LPXTG, LysM, the duplicated CW_1 and SH3 domains promoted significant surface display of two model proteins, B domain and DARPin I07, although the display achieved was lower than that for the reference anchoring domain, cAcmA. On the other hand, the ChW-containing anchoring domain of the lactococcal phage AM12 endolysin (cAM12) demonstrated surface display comparable to that of cAcmA. The anchoring ability of cAM12 was confirmed by enabling non-covalent heterologous anchoring of the B domain on wild-type bacteria, as well as anchoring of CXCL8-binding evasin-3, which provided potential therapeutic applicability; both were displayed to an extent comparable to that of cAcmA. We have thereby demonstrated the effective use of different protein anchoring domains in , with ChW-containing cAM12 the most promising alternative to the established approaches for surface display on .
在细菌表面展示重组蛋白是一个不断发展的研究领域，具有广泛的潜在生物技术应用。乳酸菌是进行这种表面展示的有吸引力的宿主，也是递送生物活性蛋白的有前景的载体。表面展示的重组蛋白通常通过锚定结构域锚定在细菌细胞壁上。在这里，我们研究了常用的含乳球菌赖氨酸基序（LysM）的表面锚定结构域（AcmA的C末端，即cAcmA）的替代物。我们筛选了15个属于Pfam类别LPXTG、LysM、CW_1、Cpl-7、WxL、SH3和ChW的乳球菌或噬菌体来源的锚定结构域，它们可以与细胞壁提供非共价或共价结合。LPXTG、LysM、重复的CW_1和SH3结构域促进了两种模型蛋白（B结构域和DARPin I07）的显著表面展示，尽管实现的展示低于参考锚定结构域cAcmA。另一方面，乳球菌噬菌体AM12溶菌酶的含ChW的锚定结构域（cAM12）表现出与cAcmA相当的表面展示。通过使B结构域在野生型细菌上进行非共价异源锚定以及CXCL8结合逃避素-3的锚定，证实了cAM12的锚定能力，CXCL8结合逃避素-3具有潜在的治疗适用性；两者的展示程度都与cAcmA相当。我们由此证明了在[具体内容未提及]中不同蛋白锚定结构域的有效应用，含ChW的cAM12是[具体内容未提及]表面展示既定方法最有前景的替代物。
