ANTI-FLUORESCEIN SINGLE-CHAIN ANTIGEN-BINDING PROTEIN

USING ITS PROPRIETARY PROTEIN ENGINEERING TECHNOLOGY GENEX HAS DESIGNED AND PRODUCED A NOVEL ANTIGEN-BINDING PROTEIN. THE DNA SEQUENCES FOR THE HEAVY (H) AND LIGHT (L) CHAINS OF A MONOCLONAL ANTIBODY TO BOVINE GROWTH HORMONE (BGH) HAVEBEEN CLONED AT GENEX AND MODIFIED TO ENCODE THE ANTIGEN-BINDING FRAGMENT I.E., THE FV FRAGMENT, AS A SINGLE POLYPEPTIDE CHAIN OF ABOUT 240 AMINO ACIDS. TO ACHIEVE THIS, DNA SEQUENCES WERE DESIGNED AND SYNTHESIZED THAT COULDBE USED AS LINKERS TO CONNECT THE DNA SEQUENCES OF THE ANTI-BGH VARIABLE LIGHT CHAIN FRAGMENT (VL) AND THE VARIABLEHEAVY CHAIN FRAGMENT (VH). THESE LINKERS ALLOW THE TWO VARIABLE DOMAINS TO FOLD AND ASSOCIATE INTO A MOLECULE THAT MIMICS THE ACTIVITY OF THE FV FRAGMENT AND IS CALLED A SINGLE-CHAIN ANTIGEN-BINDING PROTEIN (SCABP). THE MODIFIED DNA SEQUENCE HAS BEEN INCORPORATED INTO A PLASMID FOR EXPRESSION IN E. COLI WHERE THE SINGLE-CHAIN ANTIGEN-BINDING PROTEIN IS PRODUCED AT HIGH LEVELS IN THE FORM OF INSOLUBLE INCLUSION BODIES. THE SINGLE-CHAIN ANTIGEN-BINDING PROTEIN CAN BE RECOVERED FROM THESE INCLUSION BODIES AND RENATURED TO YIELD A PROTEIN THAT SHOWS SPECIFIC BINDING TO BGH. SEVERAL GOALS NEED TO BE MET TO ESTABLISH THE COMMERCIAL APPLICABILITY OF THIS TECHNOLOGY; (A) DETERMINE THE BINDING CONSTANT OF THE SCABP AND COMPARE IT TO THE MONOCLONAL ANTIBODY; (B) DEMONSTRATE THAT THE TECHNOLOGY IS GENERAL, I.E., DESIGN AND DETERMINE AFFINITIES FOR SCABP MOLECULES BINDING DIFFERENT ANTIGENS, AND (C) DEVELOP PROCEDURES FOR MORE EFFICIENT RENATURATION OF THE SCABP'S PRODUCED IN E. COLI. TO ACHIEVE THESE GOALS,AN SCABP AGAINST THE HAPTEN FLUORESCEIN WILL BE PRODUCED. THE BINDING OF FLUORESCEIN BY ITS ANTIBODIES IS MEASURED BY A STRAIGHTFORWARD FLUORESCENCE QUENCHING ASSAY WHICH WILL GREATLY FACILITATE DETERMINATION OF A BINDING CONSTANT AND THE RAPID MONITORING OF RENATURATION EXPERIMENTS.