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Protein engineering towards biotechnological production of bifunctional polyester beads

Atwood, Jane A., Rehm, Bernd H. A.
Biotechnology letters 2009 v.31 no.1 pp. 131-137
engineering, enzyme-linked immunosorbent assay, fluorescence, fluorescence microscopy, glycoproteins, green fluorescent protein, mice, myelin sheath, peptide mapping, protein engineering, sorting
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.