Preparation of the plasmid
1. Amplify the genes of the hexahistidine-ubiquitin (His6-Ub) tagged core OaAEP1-C247A and the cap domain by use of the following primers:
His6-Ub tagged core OaAEP1-C247A
2. Insert the PCR products into a pET28b vector by use of the Gibson Assembly and transform into DH5α E. coli. Confirm the DNA sequences by Sanger sequencing (e.g. through Eurofins Genomics).
3. Introduce the mutation D29E by use of the following primers:
4. Treat the PCR product with DpnI digest enzyme. Extracted plasmid from DNA agarose gel. Transformed the plasmid into DH5α E. coli. Confirm the DNA sequences by Sanger sequencing (e.g. through Eurofins Genomics).
5. Introduce the plasmid to Shuffle® T7 E. coli by heat-shock transformation.
6. Select a single colony from the agar plate to inoculate 10 mL of LB medium that is added with 50 μg/mL kanamycin and then incubate at 30 °C overnight (16-20h) with shaking at 220 rpm.
7. Inoculate the starter culture into a LB media that contains 50 μg/mL of kanamycin at a ratio of 1:100 (10 mL into 1 L) at 30 °C until an OD600 value of 0.6-0.7 is reached.
8. Induce gene expression by adding IPTG (0.4 mM) at 16 °C for 18 h and isolate the cell pellet by centrifugation (500 rpm/min, 15 min, 4 °C).
9. Resuspend the cell pellet from a 1.0 L culture in 20 mL of lysis buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8.0) with lysozyme (0.1 mg/mL), PMSF (35 µg/mL), DNase I (5 μg/mL)
10. Sonicate on ice (Amplitude: 39 %, 5 min, 5 s pulse ON, 15 s OFF) and subject the lysate to centrifugation (27,000g, 4 °C, 15 min).
11. Introduce the supernatant to 2 mL of Ni-NTA resin (Bio-Rad) in a gravity flow column (Bio-Rad).
12. Wash the column three times with lysis buffer that contains 10 mM imidazole (15 mL each).
13. Elute the remaining bound protein with 15 mL of lysis buffer that contains 300 mM imidazole.
14. Concentrate the eluted protein using a VIVASPIN centrifugal filter (10 kDa MWCO) (Cytiva).
15. Purify by size exclusion chromatography (Superdex 75, GE Healthcare) that had been pre-equilibrated in 50 mM sodium acetate buffer (pH 4.0), 150 mM NaCl, 0.5 mM TECP and 1 mM EDTA.
16. Combine fractions of desired protein and concentrate using a VIVASPIN centrifugal filter (10 kDa MWCO) (Cytiva).
17. Store at -80 °C with 5% (v/v) glycerol.
18. Perform SDS-PAGE analysis and estimate enzyme concentration by nanodrop UV-Vis A280 (Figure 2).
19. Check enzyme activity by performing peptide ligation assays in 100 μL reaction mixtures containing 50 mM Na2HPO4 (pH 7.0, 5 mM EDTA), supplemented with 1 mM peptide GLP, the AEP (10 or 40 nM) and peptide substrate (20 to 240 μM).
20. Perform each reaction in duplicate at 25 °C by use of the microplate reader (FLUOstar Omega).
21. Calculate reaction velocities by converting the slopes of fluorescence intensity change at 450 nm, which corresponds to the concentration increase of the ligated peptide product.
22. Fit data to equation 3 using GraphPad Prism 9.0.0 (GraphPad) to estimate the kinetic parameters (kcat and KM, Figure 3).
𝑣 = 𝑉𝑚𝑎𝑥 [𝑆]/(𝐾𝑀+[𝑆]) (3)