Cloning

Constructing of our antimicrobial peptide plasmid was described on our Experiment part page. Below are results confirming we clone eight of our insert genes successfully into pET28a vector.

After ordered eight insert gene sequences from IDT company. We excised it with restriction enzyme NdeI and XhoI from the vector IDT company provided then ligated eight of our insert genes into plasmid pET28a respectively. PCR amplification and enzyme digestion test are used to check that we get the right insert gene into plasmid pET28a. After that, we transformed the assembled plasmid into E. coli TOP10 for storage.

Fig.1a Using PCR colony and enzyme digestion test to analyze if the gene inserted is the size we expected. (a) The images of gel electrophoresis after enzyme digestion. In lane 1, the NEB 1 kb DNA ladder can be observed. Lane 2-6 is the result of restriction digestion test of our assembled plasmid with insert gene number 3,5,6,7,8 respectively. Using NdeI and XhoI restriction enzyme, 754-1033 bp long DNA fragments are seen, same as our expectation. (b) The images of gel electrophoresis after colony PCR of assembled plasmid with insert gene number 1,2,4. Lane 1 is the NEB 100bp6 DNA ladder. The primer pair used is T7 promoter and T7 terminator and the expected size is around 700-945. (c) The images of gel electrophoresis after colony PCR of assembled plasmid with insert gene number 3, which is control sequence. Lane 1 is the NEB 1 kb DNA ladder. The primer pair used is T7 promoter and T7 terminator and the expected size is around 700-945bp.

Overexpression of Protein

After confirming the pET28a vector, we took 1 μl of prepared DNA and transformed it into E. coli Novablue and E. coli BL21. Considering the colonies size in E. coli Novablue is more normal, we chose it for expressing our protein. We checked protein expression by incubating bacteria in 3 ml LB medium adding ImM IPTG for 2 h and ran the sample in 15% SDS-PAGE. The expected size of our protein is around 25-40 kDa.

According to Fig.2, we found out that bacteria contains insert gene No.3, 6, 7, 8 has protein induction apparently. Our choice of bacteria for protein purification was based on this result.

Fig.2a Transformation the plasmid DNA to E. coli Novablue for protein expression.

Fig.2b Coomassie blue staining SDS-PAGE showing protein induction of E. coli Novablue that contains eight different assembled plasmids individually. Lane 1 is the Thermo Scientific PageRuler Plus Prestained Protein Ladder 26619, and other contents is at the top of the lane. The one marked with (i) is the sample after IPTG induction. The bacteria that have protein expression, which contain the insert genes number 3,6,7,8, is framed in red.

Purification: Producing the AMPC

We started the process of protein purification after checking the protein induction. Assumed that all our protein is soluble first, we purified the supernatant after sonication and collect the pellet as sample for running SDS-PAGE. Using nickel column for protein purification, we eluted the protein with 50 mM, 100 mM ,200 mM, 400 Mm of imidazole respectively. Braford is used for testing the concentration of our protein, and we collected the protein washed out while it has enough concentration. By checking the protein size of SDS-PAGE, we confirmed if the protein collected is correct.

The SDS-PAGE analysis after Coomassie blue staining shows that except No.3 protein, other seven proteins are all insoluble or not induced. Therefore, we try to purify the protein by the method of solubilization of inclusion body.

Purification Results

Fig.3 Coomassie blue staining of Lysostaphin protein in 15% SDS-PAGE gel. Protein ladder we use is the Thermo Scientific PageRuler Plus Prestained Protein Ladder 26619, and contents of the lane is marked in the legend. Lane 3 shows that Lysostaphin protein is induced successfully. And Lane 7,8,9 shows the protein washed out at 100mM, 200mM, 400mM imidazole is same as the expected size 27.2kDa.

Fig.4 Coomassie blue staining of No.6 protein in 15% SDS-PAGE gel. Protein ladder we use is the Thermo Scientific PageRuler Plus Prestained Protein Ladder 26619, and contents of the lane is marked in the legend. Lane 1 shows that No.6 protein is all in inclusion body, so there isn’t any protein washed out in Lane 7,8,9.

According to Fig.5, we found that most of the target proteins is in the inclusion body. Therefore, we adjusted our method to solubilize the proteins in pellet by treating it with urea. First, we collected the supernatant after centrifugation for SDS-PAGE analysis to confirm the protein size. Then, we purified the protein with nickel column and elute it with 100mM, 200mM, 400mM imidazole.

Fig.5 Coomassie blue staining of No.1 protein after inclusion body solubilization in 15% SDS-PAGE gel. Protein ladder we use is the Thermo Scientific PageRuler Plus Prestained Protein Ladder 26619. Lane 1 is supernatant of Novablue after sonication. Lane 2 is supernatant of bacteria contain No.1 protein after sonication. Lane 3 is supernatant of bacteria pellet contain No.1 protein after method of solubilization of inclusion body and centrifugation. Lane 3 indicated that it does has protein No.1 protein in it, which the size is 36.55kDa.

Fig.6 Coomassie blue staining of No.6 protein after insoluble purification in 15% SDS-PAGE gel. Protein ladder we use is the Thermo Scientific PageRuler Plus Prestained Protein Ladder 26619, and contents of the lane is marked in the legend. Lane 2 and 3 show the protein expression before and after induction respectively. Protein induction is observed apparently in lane 3 and the size is around 31 kDa, which is nearly same size as expected. But we couldn’t see any band on the SDS-PAGE of the sample after elution, we speculated that is the reason maybe low concentration of protein.

In conclusion, we purified No.3 protein using soluble protein purification method and others with insoluble protein purification method. In considering the low concentration might affect antimicrobial activity, we concentrated the protein using protein concentrator before doing disc diffusion assay.

Disc Diffusion Assay

The antimicrobial effect of AMPC was tested by Disc diffusion assay. E.coli was selected as the control group of S. aureus on bacteria, in order to test the effect of V8 protease on AMPC. To make sure that protein has sufficient concentration for sterilization, we concentrated the protein and test the diameter of inhibition zone. To confirm the antimicrobial activity of AMPC, we observed the relationship between diameter change of inhibition zone and the concentration of AMPC.

Fig.7a The relationship between the diameter of the inhibition zone and the concentration of protein No.1, No.3, No.6 and No.7.

We found that the inhibition zone of protein No.3 was larger, suggesting a better antimicrobial effect. In protein No.6 and No.7, the effect of the zone of inhibition was also found, but higher concentrations than protein No.3 were be required. For protein No.1, no zone of inhibition was observed, which may also be the result of insufficient concentration.

Fig.7b The disc diffusion assay for protein No.1 No.3, No.6, No.7 on the S. aureus and E.coli plates

We found that the obvious inhibition zone can be observed in protein No.3, No.6 and No.7 on the S. aureus plate, but no inhibition zone was found on the E.coli-coated plate of the control group. Hence it can verify that the linker in AMPC is effectively cleaved by V8 protease of S. aureus and has the specificity of inhibiting the growth of S. aureus.

Fig.7c Antimicrobial effect of No.7 on the S. aureus plate.

In the inhibition zone of the S. aureus plate, protein No.7 was tested for multiple times. After concentrating to a much high concentration, we can see a more obvious inhibition zone appeared.