The overall goal of the following experiment is to use a multi target approach for structured determination of a target protein by x-ray crystallography, starting with just a single gene or genetic sequence in the case presented here. The target protein is the PB two polymerase subunit from influenza A, a key component of viral replication. The multi targett approach is achieved by first designing and cloning a series of genetic constructs in parallel based on the single target sequence and other information known about the protein.
The second step is to test expression levels for each construct and select the best ones for large scale protein expression in cell culture. The next step requires breaking open cells to remove the target proteins from the expression material and refining each of them to very high purity. A series of crystallization trials are then set up with each protein to obtain a crystal form suitable for x-ray studies.
High resolution x-ray diffraction data is then collected on one or more crystals and used to solve and refine an electron density map outlining the structure of each target protein. These results allow the rendering of a three dimensional structure of the target protein based on the electron density map. Multi targett processing greatly increases the likelihood of success in structured determination by providing viable alternatives at every potential roadblock in the pathway.
Working multiple targets in parallel is also highly efficient, reducing the time and cost per protein at each step. The potential of increasing the rate at which structured determination is completed for druggable targets will allow more therapy development opportunities per year. Though this method can provide valuable insight in structural biology, the high quality protein obtained through these methods can support any protein based investigation.
Each phase of the process has its own science and requires its own expertise, but the investment in people instrumentation and know-how can pay off beautifully once all the pieces are assembled. Once a target gene and gene product are selected for investigation, the first step requires design of multiple genetic variants or constructs. Gene composer software is used to design these constructs at the protein level along with the associated code on engineered synthetic gene sequences.
Using the alignment viewer module and construct design module, compare protein sequence alignments and define protein constructs design insert PCR and vector PCR terminal primers or ampers. Next, use gene composer's protein to DNA algorithm to back translate each amino acid sequence into a code on engineered nucleic acid sequence. Use the proper code on usage table to optimize the sequence for expression in a specific cell line.
In this case, e coli bacteria. Once the target gene sequence is ready, virtually clone and insert the gene into a suitable vector plasmid for bacterial expression. In this case a modified PET 28 vector.
This vector contains certain components necessary for expression and protein purification. The target gene is modified to incorporate a histamine tag sequence at either the N or C terminus of the protein and a cleavage sequence to allow removal of the tag during purification. The vector also possesses an antibiotic resistance gene for expression testing and fermentation.
Each target protein genetic sequence is then created by standard gene synthesis methods in preparation for pipe cloning polymerase incomplete primary extension cloning, or pipe cloning is a PCR based cloning strategy where the target gene is amplified with primers that are complimentary to the intended vector. This method exploits the incomplete nature of late stage PCR, which leaves the PCR products with variable single stranded termini. I prepare the insert PCR or IPCR by adding five microliters each of forward and reverse primers to each reaction.
In a 96 well plate according to a plate map, add two microliters of each full length synthetic gene to its appropriate well according to the plate map. After adding 25 microliters of PFU master mix to each, well cycle the reactions 25 times using the following PCR conditions. Denature at 95 degrees Celsius for 30 seconds.
Anil at 50 degrees Celsius for 45 seconds and extend at 68 degrees Celsius for three minutes. Fragment amplification is confirmed by AGROS gel analysis of IPCR products. Successful IPCR products are represented by robust bands.
Less desirable results are often seen as faint or smear bands in a separate reaction. The expression plasmid is amplified by vector PCR or VPCR fragment amplification is confirmed by AROS gel analysis of VPCR products. Once the IPCR and VPCR products are amplified, they're added to a merge plate and put into a thermocycler for the merge reaction.
The successfully cloned constructs are then transformed into chemically competent e coli cells and stored in glycerol stalks to begin the expression testing streak. A small amount of each sample from a glycerol stock of cloned construct onto a separate agar plate. The plate is made with bacterial nutrient broth and the antibiotic marker Kanamycin encoded in the vector incubate overnight at 37 degrees Celsius on the following day.
Start a pre culture for each sample by picking a single colony from the freshly grown agar plate. Use this colony to inoculate 1.2 milliliters of TB broth supplemented with can mycin and glucose for parallel processing. Each pre culture is grown in a 96 well round bottom block.
Grow overnight at 37 degrees Celsius with shaking at 220 rotations per minute. After the overnight growth. Start small scale induction cultures for each sample by using 40 microliters of the pre culture to inoculate.
1.2 milliliters of TB broth. Supplemented with 50 milligrams per milliliter of can mycin and novagen overnight express system. One, grow the induction cultures at 20 degrees Celsius for 48 hours, shaking at 220 RPM harvest cells by centrifugation at 4, 000 GForce units for 15 minutes.
Pour off the supernatant and store the pellets at negative 20 degrees Celsius for at least one hour prior to further processing after purification. Analyze the protein with and without cleavage reaction by capillary electrophoresis using a caliper lab CHIP 90 system according to the manufacturer's protocol. In the case of the influenza a PB two protein subunit 14 of the 34 constructs led to soluble target protein and entered the next step large scale fermentation.
To begin a large scale fermentation inoculate 100 milliliters of bacterial cell culture broth containing 50 milligrams per milliliter. Can mycin from a glycerol stock and grow overnight at 37 degrees Celsius? Shaking at 220 RPM on the following day, expand the pre culture by using 10 milliliters to inoculate.
One liter of broth containing auto induction media and 50 milligrams per milliliter can mycin in a two liter baffled flask. Shake the expanded cultures at 37 degrees Celsius approximately every 30 minutes. Check the optical density of the culture by measuring UV absorbance at a wavelength of 600 nanometers.
When an optical density of 0.6 is reached, change the temperature of the shaking incubator to 20 degrees Celsius after the desired growth time. Remove a representative 10 milliliter Ali quad from each construct for expression testing. Harvest cells by centrifugation at 5, 000 GForce units for 15 minutes.
Pour off the supernatant and freeze the cell pellet at negative 80 degrees Celsius. To begin this procedure, thaw and resuspend the cell paste in lysis buffer at a one to five master volume ratio. Vigorously stir for 30 minutes at four degrees Celsius.
Use a clean spatula to break chunks loose from the side of the beaker. Mechanically lyce the cells on ice with a masonic's sonicate. Clarify the crude lysate by centrifugation at 18, 000 GForce units for 30 minutes at four degrees Celsius.
Collect the supernatant and save a small aliquot for analysis before proceeding with large scale purification. Confirm large scale protein expression of each culture via SDS page gel analysis. This representative SDS page result shows robust protein expression, roughly 50%solubility and about 50%cleavage.
In this case, the histidine tag was removed along with an added protein solubility tag, which was engineered in the original construct. Nickel resin is used to separate the target protein with the unique histamine tag from all other cellular material including native bacterial proteins. A nickel one column is prepared by washing with four column volumes of UE water to remove storage buffer followed by one column volume of buffer B and five column volumes of buffer.
A for E equilibration parallelization is done with the protein maker, which is capable of running multiple columns at once. Load each clarified lysate containing solubilized protein with histamine tag into a separate column at a rate of two milliliters per minute, followed by several column volume washes with buffer A, collect the flow through buffer and save for analysis. Elute the bound protein in a series of step gradients with buffers A and B at 95 to five 60 to 40, and finally 100%buffer B.The components in buffer B compete with histamine for the nickel resin and thus remove the protein from the column at a given ratio.
Collect each elution fraction separately. A representative chromatogram result from a run on a nickel one column is shown here. Analyze the nickel one eluded fractions, the crude lysate, the clarified lysate, and the nickel one flow through by SDS page and compare with the UV absorbance at 280 nanometers collected during the column purification.
This step determines if the purification was successful. Select and pool fractions containing the target protein to carry forward for further processing. Calculate the total amount of protein at this stage with the theoretical extinction coefficient of the protein, measuring the UV absorbance at 280 nanometers and factoring in the total volume of the pooled fractions.
Save a small sample for analysis. The gene for the target protein was encoded with a specific sequence, which is recognized by ULP one as a cleavage site, thus adding ULP one results in cleavage between the histidine tag and the protein of interest. To begin this procedure, add one microliter of ubiquitin like protease, one for every five milligrams of protein present in the pooled fractions.
At this point, the cleaved protein is transferred from buffer B to buffer A for further processing using dialysis tubing, dialyzed the protein against two liters of buffer, A for four hours at four degrees Celsius with stirring. Use a 10 kilodalton molecular weight cutoff for PB two after dialysis. Run another SDS page gel of the target protein with and without ULP one present.
This will determine if ULP one cleavage was successful and allow selection of the best fractions to carry forward for further processing. Shown here our representative SDS page results for three constructs of the polymerase pb. Two subunit molecular weight markers are in lane one.
Lanes two, six and 10 are pooled proteins from nickel one columns. Lanes three, seven, and 11 contain flow through of cleaved protein in buffer A from nickel two and lanes four, eight and 12. Show removal of the histamine tag in buffer B from nickel two.
After removal of the histamine, the pooled fractions from nickel two are concentrated. To begin SEC, the proper resin must be chosen again based on the molecular weight of the target In this case, acere S 110 over 30 GL column is used and equilibrated with 200 milliliters SEC buffer at a flow rate of 0.5 milliliters per minute using a five milliliter syringe, load the sample into 10 milliliters sample loop and begin the SEC column run. Monitor the UV absorbent chromatogram at 280 nanometers while collecting small volume fractions.
Run all relevant SEC fractions via SDS page. The standard approach to protein crystallization trials is conducted by the sitting drop method involving vapor diffusion. It is not uncommon to set up two or more sparse matrix screens at the very outset for each target protein.
To start this process prefill each reservoir of a 96 well compact junior crystallization plate with 80 microliters each condition in the selected crystallization screen dispense 0.4 microliters of protein in SEC buffer into each of the 96 wells. Then add 0.4 microliters of the crystallization screen, ensuring complete mixing of the drop in each. Well cover the entire trite with crystal clear ceiling tape, ensuring a full seal on every well store the plate at 16 degrees Celsius in an undisturbed location free of environmental vibrations.
Check for protein crystallization periodically over the next few weeks under a dissecting microscope. If protein crystals do not appear, set new plates with different conditions and vary the protein concentration in the final drop to increase the likelihood of success before harvesting. Cool down an A LS style puck in a doer filled with liquid nitrogen and cover with a lid to begin harvesting.
Cut the clear tape covering the well with the target protein crystal to a nearby empty. Well add 1.6 microliters of the corresponding crystallizing condition and combine it with 0.4 microliters of ethylene glycol. A solution of 20%ethylene glycol in the crystallizing condition protects the protein crystal.
During cryo freezing, analyze the crystal under the microscope and select a suitable cryo loop for harvesting, matching the inner diameter of the loop to the maximum width of the crystal. Attach the cryo loop to a magnetic crystal wand and loop the crystal directly from the well solution. Immediately dip the cryo loop with the harvested crystal into a cryoprotectant and then submerge in the a LS style puck.
To flash, freeze the crystal repeat for a desired number of crystals. Once harvesting is complete, use a puck wand to place a magnetic lid on the puck containing flash frozen crystals with bent tongues. Flip the puck upside down for the next step.
Screw a puck pusher onto the puck, then transfer the puck to KU actor doer and use it to punch off the lid. The crystals in the puck remain frozen in a liquid nitrogen bath until ready for x-ray diffraction studies using the J Director image acquisition software. Set up the following parameters for crystal screening beam slit at 0.5 degrees detector distance of 50 millimeters image step at 70 degrees and exposure length of 30 seconds.
Here is an example screenshot from the image acquisition software while a crystal is being screened, the center panel shows observed x-ray diffraction from a representative crystal collect sufficient images at high resolution for a complete data set needed for three dimensional structure determination. The cloning and protein purification strategies demonstrated in this video resulted in 14 purified PB two samples from which nine yielded crystals suitable for diffraction studies. The in-house x-ray diffraction dataset was collected on five of the nine constructs with QK alpha radiation using a rigaku super bright FRE plus rotating anode x-ray generator, equipped with ossec variax hf, HF optics, and a Saturn 944 plus CCD detector.
An X-ray diffraction image of the polymerase PB two subunit is shown here. Each dataset was processed and a total of four structures of the PB two subunit were determined. Each structure was peer reviewed and uploaded to the protein data bank for public access.
This figure shows ribbon diagrams of the molecules in the crystallographic asymmetric unit. Of the four PB two structures, secondary structures are colored in a rainbow pattern with corresponding PDB codes. Shown in this table is the outcome analysis for the influenza PB two targets by the methods described in this video article.
The multi targett parallel processing pipeline for gene to structure determination is illustrated in five steps, cloning, solubility, purification, crystallization, and structure determination. The output of our structural biology pipeline not only provides the basis for structure-based research, but also feeds additional studies such as functional assays to confirm protein function or biophysical screening of novel compounds by in a MAR or SPR to identify novel starting points for drug development. This technique and the tools invented along with it have helped pave the way for structural biologists to detect many kinds of discovery-based investigations including structure-based drug design, which has had a huge impact on human health and disease.
After watching this video, you should have a good understanding of how multi targett parallel processing can greatly increase the success of structured determination. Don't forget that working in a structural biology lab can be extremely hazardous and the proper precautions, such as the use of PPE should always be taken while engaging in laboratory activities.
