Identification and monitoring of protease activity in recombinant Saccharomyces cerevisiae.
An assay for the detection of yeast (Saccharomyces cerevisiae) protease activity, using partially purified yeast-derived recombinant hepatitis B surface antigen (rHBsAg) as substrate, was developed to monitor proteolysis of rHBsAg that may occur through fermentation and isolation. The method consists of incubating small amounts of yeast lysate (protease source) with the substrate at 35 degrees C for about 16 h. Substrate proteolysis is assessed by subjecting the incubation mixtures to SDS-PAGE followed by silver-staining. The type of protease responsible for particular cleavages can be identified by treating the yeast lysates with specific protease inhibitors prior to incubation with substrate. The treatment of lysates with PMSF indicated that while many lysates possessed only serine protease activity (Protease B), some possessed proteolytic activity that could not be quenched with high levels of PMSF or other serine protease inhibitors. The use of the aspartyl protease inhibitor Pepstatin A in conjunction with PMSF virtually eliminated all proteolytic activity in these lysates, indicating that an aspartyl protease (Protease A) is expressed under some fermentation conditions. The relative amount of each protease in a lysate can be determined semiquantitatively by scanning the SDS gels densitometrically and plotting the ratio of degradates to intact antigen in the presence and absence of protease inhibitors. This method was used successfully to monitor the time-dependent expression of these proteases throughout production-scale fermentations. The impact of fermentation and purification changes on those proteases specifically responsible for the rHBsAg degradation can be easily evaluated.
Gimenez
J A
JA
Bioprocess & Bioanalytical Research, West Point, Pennsylvania 19486, USA. juan_gimenez@merck.com
Monkovic
D D
DD
Dekleva
M L
ML
D016282:Aspartic Acid Endopeptidases / Q000032:analysis / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D010450:Endopeptidases / Q000032:analysis / Q000378:metabolism*; D004789:Enzyme Activation; D005285:Fermentation; D005656:Fungal Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D006514:Hepatitis B Surface Antigens / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008828:Microbiological Techniques; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D012697:Serine Endopeptidases / Q000032:analysis / Q000378:metabolism
D005656:Fungal Proteins; D006514:Hepatitis B Surface Antigens; D011994:Recombinant Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; D016282:Aspartic Acid Endopeptidases