| Protein-ligand binding is an essential step in most biological processes and cellular functions. Molecular recognition of binding proteins has been studied in many fields of research and there have been continuous efforts toward exploiting and engineering binding proteins to develop robust, cost-effective, and highly efficient biological processes in biotechnology. Inspired by naturally evolved binding proteins that recognize different ligands with high affinity and specificity and systematically organized protein modules that control cellular functions, we have synthetically engineered binding proteins to create new biomolecules that can be applied in biosensors and biocatalysis. The first objective was to generate new binding proteins with desired functions for biosensor applications. Among the variety of binding proteins, many studies have focused on antibodies due to their unique specificity and high affinity, as well as their central role in human diseases and various utilizations as a molecular biological tool. However, there is a constant demand to create novel antibodies that can bind different targets and those with improved affinity and selectivity. In the first chapter, a new screening approach was proposed to generate novel antibodies which can be applied on the displacement format of immunosensor to detect a small molecule target. From a yeast surface display library, we screened single chain antibody fragments (scFvs) that showed good displacement capacities in the presence of the target analyte, atrazine, a major herbicide in the agriculture. After several rounds of screening using fluorescence activated cell sorter (FACS), the newly screened scFv, ATnew, demonstrated stable binding to simazine, an analog of atrazine, but displacement in the presence of atrazine. This study led to a new screening scheme to develop recombinant antibodies which possess the desired capability with the required sensitivity for small molecule detection. The second objective was to create protein architectures for more efficient biological processes. A simple and highly sensitive protein complementation-based whole-cell bioassay was developed to detect estrogenic compounds. Two split fragments of yeast cytosine deaminase (yCD) were each fused to the estrogen receptor &agr; ligand binding domain (ER-LBD) and the conformational specific peptide, &agr;beta/I. The binding of the &agr;beta/I to the ligand bound structure of ER-LBD resulted in the reconstitution of yCD. Recovered yCD activity was monitored in a life and death assay with the prodrug, 5-FC. The growth inhibition was proportional to the concentrations of 17-beta estradiol, an agonistic compound, with a very low detection limit (sub-picomolar). The designed protein pairs could be used for not only monitoring and characterizing estrogenic compounds, but also screening drugs with high sensitivity. The next focus was on constructing nanoscale protein complexes to catalyze a cascade reaction. We demonstrated a general approach for constructing a self-assembled nanoscale enzyme cascade by using engineered bacterial outer membrane vesicles (OMVs), a natural biocompartment of Gram-negative bacteria with 20 to 200 nm in diameter. Protein scaffolds containing three divergent cohesin domains for the position-specific presentation of a three-enzyme cascade were displayed on OMVs through a truncated ice nucleation protein anchoring motif (INP). The successful incorporation of the protein scaffold into the bionanoparticles and the positional assembly of three essential enzymes for complete cellulose hydrolysis to glucose were demonstrated. Glucose production from the enzyme decorated engineered OMVs was 20-fold higher than the same amount of unassembled enzymes because of the enhanced synergy among the enzymes. This result indicated the feasibility of generating bionanocatalysts for multi-enzymatic reactions in a synergistic manner by employing protein scaffolding. |
