Difference between revisions of "Part:BBa K2933001"

Revision as of 14:07, 4 September 2019

subclass B1 metallo-beta-lactamase NDM-23, codon optimized in E. coli

This part encodes a protein called NDM-23, which is a metallo-beta-lactamase of subclass B1.

Sequence and Features

Usage and Biology

NDM-23 is a type of subclass B metal beta-lactamases, which is derived from NDM-1 mutation. The beta lactamases of the NDM family can hydrolyze almost all available beta lactam antibiotics (except aztreonam) clinically, including the broad-spectrum antibiotic carbapenems. Because of the extensive substrate profile of this enzyme, the clinical strains carrying it become a great threat to human life and health.

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 use 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

Determination of the buffer composition

Determination of substrate concentration

Determination of the mikkelsen curve

Hydrolysis efficiency of different antibiotics

Inhibition of different inhibitors