Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

This protocol addresses two important aspects, characterization of an essential integral membrane, apolipoprotein acyltransferase of bacteria, and development of the assay in HDS format to screen for inhibitors of the enzyme. The method is very sensitive and suitable for screening purposes. It can contribute to the development of potential novel antibiotics.

This video will help highlight critical steps of this protocol, such as mixing, vortexing, sonicating, while working with nonhomogeneous lipid and detergent containing suspensions. Demonstrating the procedure will be Karine Nozeret, Aurelia Pernin, both are fellows in my group. Begin by preparing the reagent mixture for the apolipoprotein and acyltransferase or LNT assay.

Mix alkyne POPE and FSL-1-biotin with LNT reaction buffer in 1.5 milliliter tubes at a final volume of 18 microliters. Prepare triplicate reagent mixtures for all tested conditions. Sonicate the reagent mixtures for three minutes in an ultrasonic water bath, and incubate them at 37 degrees Celsius for five minutes.

Then add active or inactive LNT to a final concentration of one nanogram per microliter, and pipette up and down to mix. Incubate the reaction at 37 degrees Celsius for 16 hours in a thermal mixer with a heated lid. Meanwhile, prepare a stock solution of two milligram per milliliter streptavidin.

And dilute it to 10 micrograms per milliliter to create a working solution. Add 100 microliters of the solution to each well of a 96 well plate. Then air dry the wells overnight by incubating the plate at 37 degrees Celsius without a lid.

On the next day, prepare stock solutions of five millimolar Azido-FAM, 50 millimolar TCEP, two millimolar TBTA, and 50 millimolar copper sulfate pentahydrate. Perform click chemistry by adding 0.2 microliters of stock Azido-FAM, 0.4 microliters of stock TCEP, and 0.2 microliters of stock TBTA to the 1.5 milliliter LNT reaction tubes. Vortex the solution for five seconds.

Then add 0.4 microliters of stock copper sulfate. Vortex for another five seconds and incubate the reaction for one hour at room temperature in the dark. While the click chemistry mixture is incubating, wash the wells of the streptavidin coated plates three times with 200 microliters of PBST1.

Transfer 18 microliters of the click chemistry mixture to a streptavidin coated well, and add 100 microliters of PBST1 to bind the N-acyl-FSL-1-biotin FAM product to the plate. Add biotin fluorescein to the plate as a positive control for streptavidin binding and fluorescence readout. Then incubate the plate at room temperature for one hour in the dark, while shaking at 300 RPM.

After the incubation use a multi-channel electronic pipette to manually wash the plate six times with 200 microliters of PBST2. Flush it three times with the pipette. Wash it three times with 200 microliters of PBS and flush it three more times.

Add 200 microliters of PBS to each well, and detect the fluorescence at 520 nanometers in a microplate reader. Save all results in a spreadsheet, then calculate the standard deviation for each reaction as well as the P-values for negative controls and positive samples with an unpaired T test. The in vitro LNT assay described here uses phospholipids containing an alkyne fatty acid and FSL-1-biotin as substrates to form alkyne FSL-1-biotin.

Upon a click chemistry reaction with Azido-FAM, this product becomes fluorescently labeled and detectable by fluorescence spectrometry. The reaction conditions were optimized for maximum fluorescence readout at 520 nanometers. Negative controls included reactions without enzyme, with an inactive variant of the enzyme, or with thiol specific inhibitor, MTSES.

Biotin fluorescein was used as an internal control for maximum fluorescence signal. In order to develop a high throughput screening assay, the reactions were performed directly in 384 well plates. Wash steps were automated and quantity of reagents was reduced.

The reaction was sensitive and reproducible with a significant difference in fluorescent signal between the negative control and active enzyme. For high throughput screening the Z prime factor determines whether a response in an assay is large enough for screening purposes. The average Z prime factor was greater than 0.6 for the LNT assay performed in a 384 well format, suggesting that it was outstanding.

The tube assay in this video has been developed in a multi plate format that is compatible with HDS. It can be used to study other acyltransferases from a variety of organisms.