High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

The overall goal of the following procedure is to use a high throughput expression screening protocol to quantify in e coli the level of soluble proteins using an automated liquid handling robot. This is accomplished by first inoculating.96. Well pre cultures with cells transformed with plasmids encoding the target fusion proteins the next day, 24 well plates filled with auto induction media are inoculated with the overnight pre cultures to generate expression.

Cultures after harvest and freezing soluble proteins from the expression cultures are then purified by immobilized metal affinity chromatography. Ultimately, the solubility levels for each intact target fusion can be measured to determine the optimal expression conditions for protein production and the generation of successful targets. From an advantages of this technique of traditional methods of increased efficiency, the limited batch to batch radiation and the simplicity of the data handling and tracking Visual demonstration of this method is critical.

The automated steps can seem daunting and difficult to comprehend in text format alone as the text does not convey the speediness or simplicity of the procedure. After transformation of the bacteria begin by using the robotic gripper to set aside the lid of the first 24 well LB agar plate. Next, use the eight channel liquid handling arm to mix then aspirate 50 microliters of transformation mix from a 96 well transformation plate.

Dispense the transformation mix within the first four channels of the liquid handling arm into the first column of the LB agar plate and the transformations within the last four channels into the second column of the LB agar plate Wash all the tips thoroughly after dispensing and then continue transferring the transformation mix from the 96 well plate into all of the wells in the next four columns of the LB agar plate until that plate is finished. Once the transfer to the first plate has been completed, replace the lid and begin the transfer for the next plate. Once all of the transformations have been plated, shake all of the plates for one minute at 1200 RPM to generate a homogenous distribution of the transformation mix.

Then after mixing, use the 96 multichannel arm to aspirate 60 microliters of the remaining transformation mix from the 96 well plate and dispense the mix into a deep well 96 plate containing LB broth. Seal the deep well 96 pre cultures with breathable adhesive film to allow culture airation aeration, and then place the plate in a 37 degree Celsius shaking incubator at maximum speed overnight. Once the pre cultures are in the incubator, drive the LB agar plates in a hood with their lids off for about 10 minutes.

Then invert the plates in a 37 degree Celsius plate incubator overnight. The next day, use the liquid handling arm to aspirate 100 microliters of the overnight pre culture into a deep well 96 plate to inoculate the expression cultures at a one 40th dilution, using the same scheme as before, washing the liquid handling arm thoroughly at the wash station between each column of the pre cultures. When the inoculation is finished, place the expression cultures in a 37 degree Celsius shaking incubator, sealed with breathable adhesive for four hours.

Then reduce the temperature to 17 degrees Celsius for an overnight incubation. The following morning centrifuge the deep well 24 plates for 10 minutes at 3000 Gs.Discard the supernatant into a waste container containing an antimicrobial agent, and then tap the plates upside down onto absorbent paper to remove any residual medium to verify that the cultures have grown correctly. Check for the presence of pellets in the deep well.

Next aspirate 125 microliters of lysis buffer, and dispense the buffer twice into each well of the deep well 24 plates with four tips into each well. To reach the final volume of one milliliter of lysis buffer to resuspend the pellets, shake the plates at 1200 RPM for five minutes. On the robot, transfer the cell suspensions back to the appropriate wells of a deep well 96 plate and store the plates sealed at negative 80 degrees Celsius for a minimum of one hour.

For this demonstration, we will use the protocol with 50 microliters of nickel beads for purification of the target fusion proteins. First thaw the deep well 96 plate in a water bath for approximately 15 minutes. Then resus suspend the cell lysates in the shaking incubator at maximum speed for an additional 10 minutes, the cultures should become viscous.

Next, use a manual eight channel pipette to dispense DNAs and magnesium sulfate Mix into each well of the deep well 96 plate to a final concentration of 10 micrograms per milliliter and 20 millimolar respectively. Shake the plate sealed for another 15 minutes at which time the culture should become non viscous. To avoid clogging of the filter plate during the purification, it is critical to carefully visually check that none of the lysates are viscous anymore.

On the liquid handling robot, use 200 microliter wide bore tips to thoroughly mix the resin slurry before transferring 200 microliters of the slurry into the deep well 96 plate containing the lysate. Shake the deep well 96 plate at 1400 RPM and room temperature for 10 minutes to allow binding, and then use the wide bore tips to mix and then transfer the full 1200 microliters of resin lysate slurry in 200 microliter aliquots onto the filter plate. Now turn on the vacuum for approximately 30 seconds to filter the lysate through the plate, collecting the flow through in a deep well.

96 below the deep well 96 containing the flow through is removed from below the filter plate and stored on a rack until the end of the experiment. Then wash the resin twice with 800 microliters of binding buffer each time after the second wash, use the robotic gripper to place a fresh, deep, well 96 plate under the filter plate to collect the 50 millimolar I midaz wash. Add 150 microliters of wash buffer onto the filter plate and apply the vacuum again until the buffer has passed through the deep well 96 containing.

The wash is removed from below the filter plate and stored on a rack until the end of the experiment. Then after washing two more times with 800 microliters of wash buffer as demonstrated for the binding buffer washes used the robotic gripper to transfer the microplate onto the work table and put the SPE block and filter plate back on top of the microplate to collect the elucian. Then add 190 microliters of Elucian buffer to the resin in the filter plate.

After three minutes, apply the vacuum one minute to allow all of the buffer passing through. Quickly, visually check that the Ellucian volumes are correct. Finally, block samples of the Ellucian wash and flow through plates for the quantification of the level of soluble expression.

Analyze first the ellucian plate and only when necessary, check the relevant wash and flow through fractions. These data illustrate an electrophoresis result from the caliper lab chip system. The intact cleaved fusion proteins are represented by the upper bands with the cleaved protein fragments represented as the lower bands.

The fusion yields for each target protein were determined to fall within the ranges 0.1 to two, two to 10, and 10 to 25 micrograms per liter culture levels, or in some instances were not detected. Assessing the success of the protein expression by the number of diss sulfide bonds present within each fusion protein fragment demonstrates a reasonable success for all the numbers of disulfide bonds tested with the lowest success level being 66%for targets containing six disulfide bonds. Analysis of the distribution of expression success based on the isoelectric point and number of residues indicates no particular bias for the technique with both successfully expressed targets and targets that were not detected scattered throughout the plot.

Once the fusions have been detected and cleaved, the purified targets undergo quality control by electrospray ionization, mass spectrometry. Here the representative target shown is a 5.7 kilodalton dis sulfide rich venom protein with four disulfide bonds. This spectrum shows the results for the protein prior to reduction with DTT as it was after cleavage and desalting without further intervention.

This spectrum shows the protein after reduction with DTT followed by Desalting to remove any excess DTT. The arrows indicate the ions corresponding to the experimental masses and the designation for each ion is indicated in green. The experimental parent masses calculated for these ions are shown in the table and correspond to a mass difference of eight Daltons, equivalent to four oxidized die sulfide bonds.

When set up, the full protocol can be performed on more than a thousand cultures within a week. After watching this video, you should have a good understanding of how to use small scale e coli cultures to identify the successful conditions necessary for soluble protein production using high throughput methods.