@article{wilcox_clare_valentine_swaisgood_2002, title={Immobilization and utilization of the recombinant fusion proteins trypsin-streptavidin and streptavidin-transglutaminase for modification of whey protein isolate functionality}, volume={50}, ISSN={["1520-5118"]}, DOI={10.1021/jf011603c}, abstractNote={A method was developed for the production of a hydrolyzed/polymerized whey protein derivative with altered solution and gelation properties using a combination of recombinant DNA and immobilized enzyme technologies. The recombinant fusion proteins trypsin-streptavidin (TrypSA) and streptavidin-transglutaminase (cSAcTG) were produced in Escherichia coli, extracted, and then immobilized by selective adsorption on biotinylated controlled-pore glass. Recirculation through a TrypSA reactor induced limited proteolysis of whey proteins. Hydrolysates were then recirculated through a cSAcTG reactor for incremental periods of time to arrive at increasing degrees of polymerization. The polymers were subsequently analyzed for viscosity/flow behavior, gelation properties, and fracture properties using shear rate ramps/intrinsic viscosity, small-strain oscillatory rheology, and vane viscometry, respectively. By combining limited proteolysis with controlled cross-linking, it was possible to create derivatives of whey proteins with enhanced functional properties. Increases in the degree of whey protein modification were correlated with greater apparent viscosity and intrinsic viscosity, lowered gel point temperatures, and stronger, more brittle gels. This method allowed for recycling of the enzyme, eliminated the requirement for a downstream inactivation step, and permitted control over the extent of modification. Utilization of a similar process may allow for the production of designer proteins engineered with specific functionalities.}, number={13}, journal={JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY}, author={Wilcox, CP and Clare, DA and Valentine, VW and Swaisgood, HE}, year={2002}, month={Jun}, pages={3723–3730} }
 @article{clare_valentine_catignani_swaisgood_2001, title={Molecular design, expression, and affinity immobilization of a trypsin-streptavidin fusion protein}, volume={28}, ISSN={["1879-0909"]}, DOI={10.1016/S0141-0229(00)00361-6}, abstractNote={A trypsin-streptavidin (TRYPSA) fusion protein was designed and its expression in Escherichia coli was evaluated. The streptavidin gene was PCR modified and cloned into the pET expression vector. The trypsin gene was subsequently inserted into this plasmid, thus generating a colinear fusion of trypsin and streptavidin genes (pTRYPSA). This engineering strategy was verified, and TRYPSA was expressed after IPTG induction using the E. coli strains, BL21(DE3) and BL21(DE3)pLysS. Standard protein fractions of the cell lysate were prepared and trypsin activity was primarily detected in the periplasmic and inclusion body fractions. Immunoblotting showed a single Western-positive band exhibiting a molecular weight of 39,000 Da. A biotinylated porous glass affinity matrix was prepared and selective adsorption resulted in a one-step purification and immobilization of TRYPSA from crude cell lysate. Trypsin activity was verified using a synthetic substrate. This enzyme bioreactor should serve as an excellent prototype for future studies that will examine the effect of limited proteolysis on functional characteristics of milk proteins, including gelling, emulsifying and foaming properties.}, number={6}, journal={ENZYME AND MICROBIAL TECHNOLOGY}, author={Clare, DA and Valentine, VW and Catignani, GL and Swaisgood, HE}, year={2001}, month={Apr}, pages={483–491} }