N glycosyl transferase AaNGT and application thereof

[0029] Embodiment 1: Preparation of N-glycosyltransferase AaNGT

[0030] 1. Construction of expression strains

[0031] Transform the plasmid synthesized by GenScript into BL21 competent cells, heat shock at 42°C for 1 min, and spread on Amp plate. Pick a single colony into a 5ml test tube culture medium at 37°C and 200rpm for 12h, and verify the plasmid. The results of plasmid agarose gel electrophoresis figure 1 .

[0032] 2. Expression and purification of AaNGT protein

[0033] Pick a single colony BL21pET45b-AaNGT into 50ml LB medium (containing 50ug / ml ampicillin Amp) and activate at 200rpm at 37°C for 12h. Then expand the culture, transfer the bacterial solution to 1L culture medium (containing 50ug / ml ampicillin Amp), incubate at 200rpm at 37°C for about 3.5h, measure the OD value, when the OD 600 When the value is 0.6, ice-bath for 20 minutes, and then add 400 μL of 0.5 M IPTG to induce protein expression (16° C. 200 rpm). After 20 hours of induction, collect the...

[0036] Example 2: Application of N-glycosyltransferase AaNGT in polypeptide glycosylation modification

[0037] 1. Determination of enzyme activity:

[0038] The substrate peptide for the determination of enzyme activity is the fluorescently labeled hexapeptide DANYTK synthesized by Nanjing GenScript Company. Under the catalysis of NGT, it reacts with UDP-Glc or UDP-Gal respectively. The reaction solution is detected by HPLC fluorescence detector. The system is shown in Table 1-3:

[0039] Table 1-3 AaNGT enzyme activity detection reaction system

[0040]

[0041] For the reaction results, see image 3 , found that AaNGT can use UDP-Glc and UDP-Gal to modify polypeptides containing glycosylation sequences, and then perform pristine analysis on the products. The mass spectrometry results are shown in Figure 4 .

[0042] 2. Determination of optimum pH:

[0043]In order to explore the optimal pH of AaNGT, we selected different pH buffers, HAc (5.0, 6.0) PBS (6.0, 7.0, 8....