This method can help answer key questions in the glycoprotein field. Providing insight into function of the protein, the role of N-linked glycans, and the mechanism and action of antibiotics targeting the glycoprotein. The main advantage of this technique is that it can be applied to any glycoprotein of interest.
Facilitating the structural and biophysical characterization of a wide range of glycoprotein targets. Demonstrating this procedure will be Hong Cui, a research project coordinator in our laboratory. To begin, prepare and transfect cells as outlined in the text protocol.
Harvest the cells by centrifugation at 6, 371 times g and four degrees Celsius for 20 minutes. Retain the supernatant and then use a 0.22 micron filter to filter it. Load the filtered supernatant at four milliliters per minute onto a Ni-NTA column in a bench top chromatography system.
After this wash the column with three to four volumes of wash buffer. Dilute the purified glycoprotein from the column using an elution buffer gradient, while collecting fractions. Pull the fractions containing the eluded peak in a centrifugal filtration device with a 10 kilodalton nominal molecular weight limit.
Then concentrate by centrifuging at 4, 000 times G and four degrees Celsius for 15 minutes or until the sample reaches a volume of 500 microliters. Inject the concentrated glycoprotein into a 500 microliter sample loop. Load the glycoprotein onto a pre-equilibrated high-performance size exclusion column on an FPLC system at four degrees Celsius while collecting fractions.
After this run an SDS-PAGE gel to identify which eluded fractions contain the glycoprotein and pull those that do. Using a centrifugal filtration device with a nominal molecular weight limit of 10 kilodaltons, concentrate pure deglycosylated ECD at 4, 000 times G and four degrees Celsius until the desired concentration is obtained. After determining the protein concentration centrifuge the sample at 12, 000 times G and four degrees Celsius for five minutes, to remove dust and other contaminants.
Next, add 80 microliters of crystallization solution from a commercial crystallization screen to each reservoir well in a 96 well sitting drop crystalization plate. Use a crystalization robot to dispense drops of protein into the wells of the crystalization plate. Using a total drop volume of 200 nanoliters at a one to one ratio of purified protein to crystalization solution.
After this, seal the plate with tape. Transfer the sealed plate into a plate Imager for inspection by visible and ultraviolet light. Identify the conditions that give initial glycoprotein crystal heads and further optimize these crystals as outlined in text protocol.
Cryoprotect the crystals by soaking them in a solution of mother liquor supplemented with 20%glycerol. Then mount the crystals in CryoLoops. Using liquid nitrogen, flash freeze the mounted crystals prior to data collection.
After producing well-diffracting crystals in a 24 well crystalization plate, prepare a stock solution of 50 millimolar ligand and 20 millimolar Tris at pH 9.0 with 150 millimolar sodium chloride. Add various concentrations of this ligand solution to the previously prepared drop containing the ECD crystals. Seal the drop for incubation time lengths ranging between five minutes and five days.
Using a light microscope, visually track the crystals to identify any possible changes in morphology. After incubation, mount the crystals using CryoLoops and cryoprotect them in mother liquor solution supplemented with 20%glycerol. To begin Bio-Layer Interferometry, prepare 50 millimeters of 1X Kinetics Buffer from 10X Kinetics Buffer as outlined in the text protocol.
Add 200 microliters of this buffer to a pre-wetting plate. Transfer six Ni-NTA biosensors to the plate and let them hydrate in the buffer for 10 minutes. Next, dilute His-tagged ECD in one milliliter of 1X Kinetics Buffer at a final concentration of 25 nanograms per microliter.
And prepare serial dilutions of the purified FAB. Now aquate the reagents in a 96 well micro plate, as seen here. Where B represents 1X Kinetics Buffer, L represents the His-tagged glycoprotein loading, the numerical entries represent the diluted FAB concentrations, and R represents the regeneration buffer.
Transfer the hydrated biosensors into wells containing 1X Kinetics Buffer for 60 seconds to baseline them. Then, load glycoprotein at a concentration of 25 nanograms per microliter for 240 seconds at 1, 000 RPM. Place the biosensors back into the wells containing 1X Kinetics Buffer for 60 seconds for a second baseline.
Transfer the sensors into the wells containing the serial dilutions of FAB for 180 seconds. After this association phase, transfer the biosensors back into 1X Kinetics Buffer for a 180 second dissociation step. Next, open the analysis software.
Under tab one, import and then select the data. Under tab two, step one data selection, choose sensor selection. Highlight the reference wells, then right click to set the reference well.
In step two, subtraction, select reference wells. After this, under step three align Y-axis, select baseline and set a time range from 0.1 seconds to 59.8 seconds. In step four select inter-step correction and align to dissociation.
Then in step five, process Savitzky-Golay Filtering and click process data. The gate to tab three. Under step two analyze with a one to one model, select association and dissociation, select global fitting and group by color.
Right click the curves, click set color and set all of the curves to the desired colors. After this, select fit curves. If the data is well fitted, export the result by clicking save report.
Repeat the experiment for all desired conditions. In this study several constructs of the CD22 extracellular domain are successfully cloned into the pHL-sec expression vector. These constructs are then over expressed in the mammalian HEK293-F and HEK293-S cell lines.
While the cultures containing approximately one million and one and a half million cells per milliliter, expressed very similar levels of glycoproteins. Cultures of only half a million cells per milliliter expressed noticeably less. After this, cultures are harvested and ECD glycoprotein is isolated by affinity and size exclusion chromatography to purify all constructs to size homogeneity.
Yielding a highly pure sample for crystalization and biophysical studies. Following these procedures, other methods like, singular particle electron microscopy can be performed in order to answer additional questions about the three dimensionalist structure of the full length extracellular domain or protein complexes. After watching this video, you should have a good understanding of how to express, purify, and structurally and biophysically characterize glycoproteins, their ligands, and targeting antibodies.
