Part:BBa_K2933223

RBS a+Linker g+GST+Linker e+NDM-23

This part consists of RBS a, protein coding sequence(GST+Linker e+NDM-23), the RBS and the protein coding sequence can be connected by linker g. The biological module can be build into E.coli for protein expression. This part can be prefaced with promoters of different strengths and types to regulate expression function.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 113
  

Usage and Biology

This composite part is made up with five basic parts, the RBSa, the linker g and the GST tag, the cutting site of Prescission Protease and our target protein NDM-23. It encodes a protein which is NDM-23 fused with GST tag. The fusion protein is about 54.5 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of NDM-23 and combine the four 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.

Origin(organism)

Klebsiella pneumoniae.

Molecular cloning

First, we used the vector pGEX-6p 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 NDM-23. 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 GST Agarose to purify the target protein. The GST Agarose can combine specifically with the GST tag fused with target protein.

• First, wash the column with GST-binding buffer for 10 minutes to balance the GST column.
  
• Second, add the protein solution to the column, let it flow naturally and bind to the column.
  
• Third, add GST-Washing buffer several times and let it flow. Take 10μl of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
  
• Forth, add 400μL Prescission Protease (1mg/mL) to the agarose. Digest for 16 hours in 4℃.
• Fifth, add GST-Elution buffer several times. Check as above. Collect the eluted proteins for further operation.
  

Figure 2. The result of SDS-page.

Anion exchange column:
According to the predicted pI of the protein and the pH of the ion-exchange column buffer, firstly select the appropriate ion exchange column (anion exchange column or cation exchange column). The pH of buffer should deviate from the isoelectric point of the protein. Since the isoelectric point of our protein is 5.88 in theory, we choose buffer pH of 7.4 and use anion exchange column for purification. The protein is concentrated with a 10KD concentration tube, and then the exchange buffer is used to exchange the protein to the ion-exchange liquid A. Finally, it is concentrated to less than 5ml by centrifuging at 4℃ and 3400rpm for 10 minutes in a high-speed centrifuge to remove insoluble substances and bubbles. Balance the selected column with liquid A. Through the AKTApure protein purification system, the samples are loaded to the column at a flow rate of 0.5ml/min, and continue washing for 5min. Gradually increase the content of liquid B in the column, change the salt concentration and then change the interaction between the sample and the column, and collect the corresponding eluent according to the position of the peak. Use SDS-PAGE to check the result.

Figure 3. The result of SDS-page of superdex75 Q column.

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 4. (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 28.5kD.

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 5. The concentration of CDC-1 was fixed at 10.5 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 6. We took the emission fluorescence at 3.02nm 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 1.648nM.

Determination of the buffer condition

Figure 7. Effect of different buffer condition on enzyme activity.

According to the experimental results, we chose NaCl concentration of 140mM, ZnCl concentration of 25 micron and pH of 8.0. The effect of DMSO on protein activity can be excluded in the range of 2-10%. (6% in the system)

Lineweaver-Burk plot

Figure 8. The relationship between the substrate concentration and the maximum initial rate was obtained by using the Lineweaver-Burk plot.

Figure 9. The relationship between the maximum fluorescence value and substrate concentration.

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 10. The enzyme kinetic parameter of NDM-23.

Establishment of NDM-23 inhibitor screening system

After the above determination of enzyme activity and the trial of concentration and buffer components, we determined the optimal conditions of NDM-23 enzyme activity and then established the screening system.

Figure11. Protein concentration and optimal buffer components of NDM-23.

Figure12. The inhibitor screening system of NDM-23.

Effective inhibitors in vitro we found

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 20 inhibitors with significant inhibitory effect on NDM-23 from the drug library containing over 4000 small molecules.

Extracorporeal IC50 and inhibitory mechanism of inhibitors

We tested the IC50 of eight inhibitors and the inhibitory mechanism of 6 inhibitors.

Figure 13. IC50 and inhibitory mechanism of Tannic acid for NDM-23. Its inhibition type is competitive inhibition of reversible inhibition.

Figure 14. IC50 and inhibitory mechanism of (-)-Epigallocatechin gallate(EGCG) for NDM-23.Its inhibition type is competitive inhibition of reversible inhibition.

Figure 15. IC50 and inhibitory mechanism of Corilagin for NDM-23.Its inhibition type is uncompetitive inhibition of reversible inhibition.

Figure 16. IC50 and inhibitory mechanism of silver sulfadiazine for NDM-23.Its inhibition type is uncompetitive inhibition of reversible inhibition.

Figure 17. IC50 and inhibitory mechanism of Fingolimod hydrochloride for NDM-23.Its inhibition type is competitive inhibition of reversible inhibition.

Figure 18. IC50 and inhibitory mechanism of ginkgoic acid for NDM-23.Its inhibition type is irreversible inhibition.

Figure 19. IC50 of Etravirine for NDM-23.

Figure 20. IC50 of Adapalene for NDM-23.

Monitoring in living bacterial cells with antibiotics

1.Experiments proving the accuracy of the system
Firstly, NDM-23 protein solution at 3.02nM was mixed with cefazolin at 250 μM, and the characteristic absorption peak of cefazolin at 273nm obtained by our experiment was continuously measured.

Figure 21. The curve of specific absorption peak of antibiotics mixed with NDM-23 was revealed.

Secondly, after phosphate buffer treatment, E. coli strain transferred to NDM-23 beta-lactamase gene was diluted to OD600=0.15, and the strain was mixed with cefazolin with a final concentration of 250 μM, and the peak value of cefazolin at 273nm was continuously measured. The results are shown below.

Figure 22.The curve of specific absorption peak of antibiotics mixed with E. coli with the NDM23 beta-lactamase gene was revealed.
At the same time, the phosphate buffer used in the preparation of bacterial solution and the E.coli without the transfer of NDM-23 beta-lactamase gene were mixed with cefazolin with a final concentration of 250 μM respectively as blank control and negative control, and the specific absorption peak value of cefazolin at 273nm was continuously determined.


Figure 23. The curve of specific absorption peak of antibiotics mixed with the phosphate buffer and the E.coli without the transfer of NDM23 beta-lactamase gene was revealed. The one up is antibiotic combined with phosphate buffer and the one down is antibiotic combined with the E coli without the transfer of NDM-23 beta-lactamase gene.

2.Hydrolysis efficiency to different antibiotics

Figure 24. The stability of four antibiotics in the presence of the NDM23 E. coli cells. The hydrolysis of antibiotics was monitored through absorbance changes of the drugs at 273, 300，307 and 360 nm for cefazolin, meropenem and faropenem, and tetracycline,respectively,in the kinetic mode (120min, triplicate scans per 5 min). The starting concentration of antibiotics was 250 μM.

3.Evaluation of therapeutic effect of NDM-23
After determining the reaction system, we diluted the concentration gradient of several inhibitors and incubated them with the prepared E.coli which has been transferred into NDM23 beta-lactamase gene in dark for 1h, then added cefazolin with a final concentration of 250 μM and continuously measured the light absorption value at 273nm. The results are as follows:

Figure 25. Monitoring in living bacterial cells with antibiotics and EDTA(positive control).

Figure 26. Monitoring in living bacterial cells with antibiotics and Corilagin.

Figure 27. Monitoring in living bacterial cells with antibiotics and EGCG.

Figure 28. Monitoring in living bacterial cells with antibiotics and Tannic acid.

Conclusion

In conclusion, NDM-23 protein was successfully expressed in this part. We measured enzyme activity, established the high-throughput screening system, successfully screened some 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 the inhibitors can effectively inhibit the activity of the enzyme in vivo and prevent the hydrolysis of cefazolin by the enzyme. We are proud that our results have laid the foundation for further research.

Reference

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