Difference between revisions of "Part:BBa K2933163"

Revision as of 11:56, 20 October 2019

Tac promoter+RBS a+Linker g+GST+Linker e+ElBlaII

This part consists of Tac promoter,RBS and protein coding sequence (GST+Linker e+ElBlaII),and the biological module can be built into E.coli for protein expression.

Sequence and Features

Usage and Biology

This composite part is made up with six basic parts(Tac promoter，RBS a , Linker g, GST, Linker e and ElBlaII). It encodes a protein which is ElBlaII fused with GST tag. The fusion protein is about 53.5 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of ElBlaII and combine the two parts with the cutting site of Prescission Protease. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pET28b-sumo to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

Figure 1. Left: The PCR result of ElblaII. Right: The verification results by enzyme digestion.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.

Expression and purification

Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.
Massive expressing:
After taking samples, we transfered them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 1 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.

Affinity Chromatography:
We used the Ni Agarose to purify the target protein. The Ni Agarose can combine specifically with the Ni-Sumo tag fused with target protein.

• First, wash the column with water for 10 minutes. Change to Ni-binding buffer for another 10 minutes and balance the Ni column.
  
• Second, add the protein solution to the column, let it flow naturally and bind to the column.
  
• Third, add Ni-Washing buffer several times and let it flow. Take 5ul of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
  
• Forth, add Ni-Elution buffer several times. Check as above.
  
• Fifth, collect the eluted proteins for further operation.
  

Gel filtration chromatography:
The collected protein samples are concentrated in a 10 KD concentrating tube at a speed of 3400 rpm and concentrated for a certain time until the sample volume is 500 μl. At the same time, the superdex 200 column is equilibrated with a buffer to balance 1.2 column volumes. The sample is then loaded and 1.5 cylinders are eluted isocratically with buffer. Determine the state of protein aggregation based on the peak position and collect protein samples based on the results of running the gel.

Figure 1. (a) The result of gel filtration used the superdex75 column with the AKTA system, which shows that the target protein is monomeric. (b) The result of SDS-PAGE. And the target protein is about 26kD.

Enzyme activity determination

We used CDC-1, a probe with a similar structure from the beta lactam ring and a luminescent group for enzyme activity measurements. For more information on the substrate CDC-1, please see our project introduction.

Materials:
General 96-well plates (Black)
Infinite M1000 Pro Automatic Microplate Reader
Multi-channel adjustable pipette
Ultrasonic Cleaner

Buffer:
100% DMSO
Fluorescent Probe(CDC-1)
Target Enzyme(beta-lactamase)

Determination of enzyme concentration

Figure 2. The concentration of CDC-1 was fixed at 8.5 μM and the enzyme concentration was changed within a certain range, and the fluorescence value was measured with a function of reaction time. Left： First, we selected three gradient concentrations (with large intervals) for pre-experiment, and determined the gradient range of the formal experiment through the experimental results. Right： The appropriate enzyme concentration was selected for determination of the gradient, and the reaction curve of gradual rise was obtained.

Figure 3. We took the emission fluorescence at 30.2nm as the maximum emission fluorescence, and took the logarithm value of different NDM-23 enzyme concentrations to make the relationship curve between protein concentration and fluorescence emission rate. When the emittance of the system was 80%, the protein concentration was 2.273nM, that is, EC80 was 2.273nM.

Determination of the buffer condition

Figure 4. Effect of different buffer condition on enzyme activity.

According to the experimental results, we chose NaCl concentration of 35mM, ZnCl concentration of 25 micron and pH of 8.5.

Michaelis-Menten plot

Figure 5. The relationship between the substrate concentration and the maximum initial rate was obtained by using the Michaelis-Menten plot.

Calculate Km, Vm with the Lineweaver-Burk plot, because it fit better. Kcat values were calculated with the results of maximum fluorescence values at different substrate concentrations.

Figure 6. The enzyme kinetic parameter of ElblaII.

Establishment of ElblaII inhibitor screening system

After the above determination of enzyme activity and the trial of concentration and buffer components, we determined the optimal conditions of Elbla2-1 enzyme activity and then established the screening system.

Figure7. Protein concentration and optimal buffer components and the inhibitor screening system of ElblaII.

Effective inhibitors in vitro we founded

Above, we have established the NDM-23 high-throughput screening system, and then we used the microplate reader to conduct high-throughput screening to screen out nearly 10 inhibitors with significant inhibitory effect on NDM-23 from the drug library containing over 4000 small molecules

IC50 and inhibitory mechanism of inhibitors

We tested the IC50 of two inhibitors.

Figure 8. IC50 of Tannic acid for ElBlaII.

Figure 9. IC50 of Isoconazole nitrate for ElBlaII.

Monitoring in living bacterial cells with antibiotics

After high-throughput screening, tannic acid was screened as the inhibitor of ElBlaII. We have used the UV visible method to assess the effectiveness of the treatment. The results are as follows:

Figure 10. Monitoring in living bacterial cells with antibiotics and Tannic acid.

Conclusion

In conclusion, beta-lactamase II [Erythrobacter litoralis HTCC2594] protein was successfully expressed in this part. We measured enzyme activity, established the high-throughput screening system, successfully screened dozens of effective inhibitors with CDC-1 probes and then verified some of them with live bacteria to determine the IC50 of the inhibitors in vivo.We found that inhibitors can effectively inhibit the activity of the enzyme in vivo and prevent the hydrolysis of cefazolin by the enzyme.The resulting inhibitors lay the foundation for future clinical use.

References

[1] Girlich D, Poirel L, Nordmann P, Diversity of naturally occurring Ambler class B metallo-β-lactamases in Erythrobacter spp. The Journal of Antimicrobial Chemotherapy [31 Jul 2012, 67(11):2661-2664]