Swee Lee Kim, Lourido Sebastian, Bell George W, Ingram Jessica R, Ploegh Hidde L
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, United States.
ACS Chem Biol. 2015 Feb 20;10(2):460-5. doi: 10.1021/cb500462t. Epub 2014 Nov 10.
Surface display of engineered proteins has many useful applications. The expression of a synthetic chimeric antigen receptor composed of an extracellular tumor-specific antibody fragment linked to a cytosolic activating motif in engineered T cells is now considered a viable approach for the treatment of leukemias. The risk of de novo tumor development, inherent in the transfer of genetically engineered cells, calls for alternative approaches for the functionalization of the lymphocyte plasma membrane. We demonstrate the conjugation of LPXTG-tagged probes and LPXTG-bearing proteins to endogenous acceptors at the plasma membrane in a single step using sortase A. We successfully conjugated biotin probes not only to mouse hematopoietic cells but also to yeast cells, 293T cells, and Toxoplasma gondii. Installation of single domain antibodies on activated CD8 T cell redirects cell-specific cytotoxicity to cells that bear the relevant antigen. Likewise, conjugation of Toxoplasma gondii with single domain antibodies targets the pathogen to cells that express the antigen recognized by these single domain antibodies. This simple and robust enzymatic approach enables engineering of the plasma membrane for research or therapy under physiological reaction conditions that ensure the viability of the modified cells.
工程蛋白的表面展示有许多有用的应用。由与工程化T细胞中的胞质激活基序相连的细胞外肿瘤特异性抗体片段组成的合成嵌合抗原受体的表达，现在被认为是治疗白血病的一种可行方法。基因工程细胞转移中固有的从头肿瘤发生风险，需要淋巴细胞质膜功能化的替代方法。我们展示了使用分选酶A在一步中将LPXTG标记的探针和携带LPXTG的蛋白质与质膜上的内源性受体结合。我们成功地将生物素探针不仅与小鼠造血细胞结合，还与酵母细胞、293T细胞和弓形虫结合。在活化的CD8 T细胞上安装单域抗体可将细胞特异性细胞毒性重定向到携带相关抗原的细胞。同样，将弓形虫与单域抗体结合可将病原体靶向表达这些单域抗体识别的抗原的细胞。这种简单而稳健的酶促方法能够在确保修饰细胞活力的生理反应条件下对质膜进行工程改造，用于研究或治疗。
