Part:BBa_K2933002

subclass B3 metallo-beta-lactamase SPG-1, codon optimized in E. coli

This part encodes a protein called SPG-1, which is a metallo-beta-lactamase of subclass B3.

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
COMPATIBLE WITH RFC[1000]

Usage and Biology

blaSPG-1, a new carbapenem-hydrolyzing beta-lactamases , is analyzed from Sphingomonas sp. in the soil microbiota. This enzyme has not yet emerged in clinical settings but constitute potential carbap- enem resistance determinants in pathogenic bacterial species, as demonstrated by their ability to confer resistance to ampi- cillin and various cephalosporins, as well as reduced suscepti- bility to carbapenems, once expressed in E. coli.

References

Molecular cloning

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 transfer 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 0.3 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask. Put the liter flasks in 16°C shaking incubator for 16h. Centrifuge your bacteria in 500 mL bottles in the 4°C rotor at 4,000 RPM for 20 mins. Do this in batches until all your culture is spun down saving the cell pastes each time.

Purification of GST fusion proteins:

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 around 5 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.

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 28kD.

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 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 3. We took the emission fluorescence at 24.72nm as the maximum emission fluorescence, and took the logarithm value of different SPG 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 21.80nM, that is, EC80 was 21.80nM.

Determination of the buffer composition

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 9.5.

Determination of substrate concentration

Figure 5. (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 6. 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 7. The enzyme kinetic parameter of SPG.

Hydrolysis efficiency of different antibiotics

We used the enzyme concentration we had determinded to make a high throughput screens. The substrate was also CDC-1.