Studies On Design Of Protease-Based Signal Amplification System

ObjectsTo develop a specific, rapid, effective protease signal amplification system. To provide a new methodology for the clinical molecular diagnosis. To design HRV3C protease signal amplification system and TEV protease signal amplification system.MethodsWe try some different strategies to construct recombinant zymogen, such as inverting the N-terminal and the C-terminal domains, inserting or deleting amino acid residues, constructing FRB-Protease-N-FPR-Protease-C tetramer, and reconstituting the N-terminal, C-terminal fragments. The cDNAs encoding recombinant zymogens carrying the cleavage site of the protease were cloned into pET24a vector and expressed in E.coli strain BL21(DE3). Through Ni column affinity chromatography purification, recombinant zymogens were obtained. We then screen the recombinant zymogens that could be used in protease-based signal amplification system using biochemical assays with serial diluted initiating protease.Results1) We constructed four series recombinant zymogens for HRV3C protease signal amplification system. The series of HRV3C-Rev contains11recombinant zymogens. The series of HRV3C’s active sites contains3recombinant zymogens. The series of deleting&inserting middle linker loop contains5recombinant zymogens. The series of HRV3C-tetra contains1recombinant zymogen, FRB-HRV N-FPR1-HRV C. HRV3C-Rev-79,80, HRV3C-43,44&HRV3C-66,67self-processed during the Ni column purification. HRV3C-Rev-106,107can self-process with time increasing. Without raprmycin, HRV3C-tetra was self-processed after longtime induction. The digestion of other recombinant zymogens were dependent on the concertration of HRV3C.2) We constructed three series recombinant zymogens for TEV protease signal amplification system. The series of TEV-Rev&deleting contains21recombinant zymogens. The series of TEV-tetra contains1recombinant zymogen, FRB-TEV N-FPR1-TEV C. The series of TEV-Rev&deleting contains21recombinant zymogens. The series of TEV N,C fragment contains2recombinant zymogens. The recombinant zymogens from series of TEV-Rev&deleting were self-processed after short-time induction, except TEV-Rev14-222. The digestion of TEV-Rev14-222recombinant zymogens was dependent on the concertration of TEV. Without raprmycin, TEV-tetra was self-processed after short-time induction. TEV-N fragment has enzyme activity. TEV-N&TEV-C could be activited while mixing them.ConclusionThe recombinant zymogens we constructed cannot satisfy the standard of protease signal amplification system we want to develop. But we find some recombinant zymogens have the ability to be the candidates.