Methods of generating and screening for proteases with altered specificity
A technology of protease and serine protease, which is applied in fusion with protease sites, biochemical equipment and methods, and screening of compounds, and can solve problems such as sequence specificity differences of large families of proteases
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specific Embodiment approach
[0134] DETAILED DESCRIPTION: Comparison of a control indicator solution lacking protease provides a measure of protease activity. By drawing a standard curve of the protease / indicator combination, in which the rate of change of fluorescence produced by the known active protease solution is measured, the activity level can be accurately quantified.
[0135] When the measurement of the fluorescent compound is preferably completed using a fluorometer, various other methods known to those skilled in the art can be used to complete the measurement. Therefore, for example, when the luminescent group emits visible light, the measurement only requires visual observation of the fluorescence in response to the excitation of the light source. The measurement can also be done through an image analysis system, using a camera coupled with a digitizer or other image acquisition system. The measurement can also be done through a filter using contrast methods, for example under a fluorescence micr...
Embodiment 1I99
[0187] Example 1. Preparation and storage of I99A granzyme B
[0188]The wild-type murine granzyme B structure was prepared as described above (Harris et al., Journal of Biochemistry, 1998, Issue 273, pages 27364-27373). The following point mutations were introduced into the pPICZαA plasmid: N218A, N218T, N218V, I99A, I99F, I99R, Y174A, Y174V. Each variant was confirmed by sequencing the 5'AOX and 3'AOX regions using primers, and then transformed into X33 cells, and then selected using Zeocin (Invitrogen, La Jolla CA). The expression and purification of each variant is the same as the aforementioned method for wild-type murine granzyme B (Harris et al., Journal of Biochemistry, 1998, Issue 273, pp. 27364-27373).
[0189] Using the QuikChange (Stratagene) method of site-targeted mutation induction, the mutation protease murine granzyme B changed the isoleucine at position 99 to alanine. The DNA primer for introducing the I99A variant is: CCA GCG TAT AAT TCT AAG ACA GCC TCC AAT GAC...
Embodiment 2
[0192] Example 2. Synthesis and Screening of ACC Location Scanning Library
[0193] Synthesis of ACC-resin
[0194] Treat 7-aminocoumarin-4-acetic acid with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) to prepare 7-Fmoc-aminocoumarin-4-acetic acid. Mix 7-aminocoumarin-4-acetic acid (10.0 g, 45.6 mmol) and water (228 mL). Then, a small portion of sodium bicarbonate (3.92 g, 45.6 mmol) was added, and then acetone (228 mL) was added. The solution was cooled using an ice bath, and 9-fluorenylmethyloxycarbonyl chloride (10.7 g, 41.5 mmol) was added during one hour of stirring. Remove the ice bath and stir the solution overnight. After the acetone was removed by rotary evaporation, the resulting viscous solid was collected by filtration, and part of the hexane was used for washing again. 7-Fmoc-aminocoumarin-4-acetic acid was used to condense Rink amide AM resin to prepare ACC-resin. Rink amide AM resin (21 g, 17 mmol) was dissolved in 200 ml of dimethylamide (DMF). The mixture was s...
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