Difference between revisions of "Part:BBa K2933123"

Revision as of 13:04, 24 September 2019

His+Linker a+Sumo+Linker b+NDM-23

This part encodes the fusion protein of His tag, sumo tag and NDM-23 to promote the expression and purification of target protein(NDM-23).

Sequence and Features

Usage and Biology

This composite part is made up with four basic parts, the His tag, the Sumo tag，three cutting sites(NdeI,NheI and BamHI) and our target protein NDM-23. It encodes a protein which is NDM-23 fused with His-Sumo tag. The fusion protein is about 40.5kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of NDM-23 and combine the two parts with three cutting sites. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pGEX-6p-1 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.
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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. (a) 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. (b) 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)

Michaelis-Menten plot and Lineweaver-Burk plot

Figure 8. (a) The relationship between the substrate concentration and the maximum initial rate was obtained by using the Michaelis-Menten plot. (b) 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.

Hydrolysis efficiency to different antibiotics

Figure11. 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 (60min, triplicate scans per 5 min). The starting concentration of antibiotics was 500 μM.

Effective inhibitors in vitro we founded