Atwood Jane A, Rehm Bernd H A
Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
Biotechnol Lett. 2009 Jan;31(1):131-7. doi: 10.1007/s10529-008-9836-9. Epub 2008 Sep 18.
Microbial polyester inclusions have previously been demonstrated to be applicable as versatile beads outside the bacterial cell. Engineering of proteins selectively binding to the polyester inclusions was conceived to produce polyester beads simultaneously displaying two protein-based functions suitable for applications in, for example, fluorescence activated cell sorting (FACS). The polyester synthase and the phasin protein were fused to the green fluorescent protein (GFP) and the murine myelin oligodendrocyte glycoprotein (MOG), respectively, or GFP and MOG were fused to the N- and C-terminus, respectively, of only the phasin. In both cases, fusion proteins were found to be attached to isolated polyester inclusions while displaying both functionalities per bead. Functionalities at the bead surface were assessed by ELISA, FACS and fluorescence microscopy. The respective double fusion protein was identified by peptide fingerprinting using MALDI-TOF/MS.
微生物聚酯内含物此前已被证明可作为细菌细胞外的多功能珠子使用。人们设想通过工程手段使蛋白质选择性地结合到聚酯内含物上，从而生产出同时展现两种基于蛋白质功能的聚酯珠子，适用于例如荧光激活细胞分选（FACS）等应用。聚酯合酶和相蛋白分别与绿色荧光蛋白（GFP）和小鼠髓鞘少突胶质细胞糖蛋白（MOG）融合，或者GFP和MOG分别仅与相蛋白的N端和C端融合。在这两种情况下，均发现融合蛋白附着于分离出的聚酯内含物上，且每个珠子都展现出两种功能。通过酶联免疫吸附测定（ELISA）、FACS和荧光显微镜对珠子表面的功能进行了评估。使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF/MS）通过肽指纹图谱鉴定了相应的双融合蛋白。
