Engineering of fluorescent antibody in bacteria

• Main Author: Okou, David
• Format: Others
• Published: DigitalCommons@Robert W. Woodruff Library, Atlanta University Center 2002
• Subjects: Engineering; fluorescent antibody in bacteria; Chemistry
• Online Access: http://digitalcommons.auctr.edu/dissertations/3222; http://digitalcommons.auctr.edu/cgi/viewcontent.cgi?article=4739&context=dissertations

Towards the construction of protein-based biological sensors, chimeric proteins comprised of an antibody single chain antigen-binding protein (scFv) and the green fluorescent protein (GFP) were constructed. Although correct folding of the scFv domain typically requires the oxidizing conditions of extracellular compartments, such as the periplasmic space of E. coli, GFP is unable to mature under these conditions. Using DNA recombinant technology, fusion constructs were made in the cytoplasm under control of the araBAD promoter. Weak fluorescence of the GFP domain and antigen binding activity of the sFv domain were obtained in the cytoplasm of E. coli BL21, but improved expression and activities of both domains were obtained by using a trxB- mutant of E. coli, as well as by modifying physical and genetic conditions for expression of the fusion proteins. Assessment of the fluorescence and antigen binding activity of the fusion proteins indicates that GFP fluorescence can serve as an indicator of correct folding of fusion proteins.