Protein engineering towards biotechnological production of bifunctional polyester beads.
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.
Atwood
Jane A
JA
Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
Rehm
Bernd H A
BH
D000595:Amino Acid Sequence; D001709:Biotechnology; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D008863:Microspheres; D008969:Molecular Sequence Data; D010799:Phycoerythrin / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011091:Polyesters / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011091:Polyesters; D011993:Recombinant Fusion Proteins; D010799:Phycoerythrin; D016650:Fluorescein-5-isothiocyanate