Genetic Engineering of Bovine κ-Casein To Enhance Proteolysis by Chymosin

k-Casein cDNA was mutated to change the chymosin sensitive site from a Phe(105)-Met(106) bond to a Phe(105)-Phe(106) bond which, in theory, should be attacked at a faster rate by acid proteases. Mutant k-casein and normal k-casein were expressed in E. coli strain AR68 using the secretion vector, pIN-III-ompA. The expressed k-caseins were extracted in urea buffer and partially purified using DE 52 anion exchange chromatography. Partially purified k-caseins were hydrolyzed with chymosin at 30°C. Initial hydrolysis rates were compared. The mutant k-casein (Phe(105)-Phe(106)) was hydrolyzed approximately 80 percent faster than the wild-type (Phe(105)-Met(106)) k-casein as determined using western blots, followed by immunochemical staining and laser gel scanning.