Design of helical proteins for real-time endoprotease assays.
Proteases play a key role in cellular biology and have become priority targets for new pharmaceuticals. Thus, there is a high demand for specific, sensitive, and quick assays to monitor the activity of endoproteases. We designed affinity-tagged helical proteins with unique protease cleavage sites and thus constructed universal, molecularly defined, and uniform substrates for in vitro detection of IgA endoprotease. The substrate is a 10.5-kDa recombinant helical protein with a high-affinity (His)(6)-tag at the amino-terminal end. Further elements are a unique proteolytic recognition site and a C-terminal helical extension, which is cut off by the protease. Proteolytic action can be monitored in real time using surface plasmon resonance spectroscopy. Femtomole amounts of protease could be reliably and quantitatively detected within a few minutes after the start of the reaction. The detection signal changed linearly with the amount of protease and was independent of the applied sample flow rate. The biochip can be reversibly loaded with the recombinant protease substrate, so that the SPR assay is well-suited for automation. By substituting an HIV protease site for the recognition site of the IgAse, we also obtained a substrate for the quantitative and sensitive detection of HIV-1 endoprotease. Our substrate design is thus generally applicable.
Steinrücke
P
P
Molecular Biology Department, Institute of Molecular Biotechnology, IMB Jena e.V., Beutenbergstrasse 11, Jena, 07745, Germany. pstein@imb-jena.de
Aldinger
U
U
Hill
O
O
Hillisch
A
A
Basch
R
R
Diekmann
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D019963:Clinical Chemistry Tests / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D016333:HIV Protease / Q000032:analysis / Q000737:chemistry; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000032:analysis / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*; D013997:Time Factors
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C021433:IgA-specific serine endopeptidase; D016333:HIV Protease