Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAΔ41

The purification process begins with cell pellets expressing the Mame. Delta 41 recombinant protein cell membrane disruption is followed by separation of the soluble and insoluble fraction. In order to extract the soluble recombinant protein, affinity chromatography separates the tagged recombinant protein from other cellular proteins.

Ion exchange chromatography separates mamma Delta 41 from other cellular proteins that remain after affinity chromatography size exclusion. Chromatography further separates mamma Delta 41 and reveals its oligomeric state. Finally, the recombinant protein purity and homogeneity is analyzed.

Hi, I'm Raza, the head of the XVA Laboratory in the Life Sciences Department at the Benon University of the Negative. I'm Natalie also from Z Lab. Today we will show you a procedure for recombinant protein purification.

We use this procedure in our laboratory to study magnetism associated proteins. So let's get started At the start of this protocol. The MAME Delta 41 protein is expressed as a 10 hiss fusion protein.

In BL 21 e coli cells grow the cells in auto induction media containing Epi Brazilin at 310 K for three hours. Then shift the cultivation temperature to 300 K and allow the cells to incubate for 48 hours. Following incubation, harvest the cells by centrifugation, decant the supernatant and weigh the resulting cell pellet.

Eight liters of culture should produce about 60 grams of wet cell pellet. Prior to protein purification. Prepare 200 milliliter stalk solutions of four molar ile, five molar sodium chloride, and one molar tris H-C-L-P-H eight.

Using double distilled water or DDW vortex the solutions until clear and filter them with a point 22 micrometer. Filter from the stock solutions freshly prepared and filter buffers A through F as described in the written protocol accompanying this video. To begin protein purification, re suspend the cells in buffer A in a one to two ratio of cell mass to buffer volume.

For every 10 grams of bacteria, add 10 microliters of dna, one to digest any DNA fragments in the sample. Then add one milliliter of E dt, a free protease inhibitor cocktail. For every 20 grams of bacteria, incubate the solution for 20 minutes on ice.

Following incubation, lyce the cells with two cycles of French press at 25, 000 PSI. Due to temperature buildup, the piston should be cooled on ice for 10 minutes prior to use. Transfer the lysed cells into 25 milliliter ultracentrifuge tubes.

Fill the tubes to the neck line with buffer A and balance them precisely. Then separate the cell debris by ultracentrifugation. Meanwhile, assemble a nickel NTA gravity column by adding four milliliters of nickel NTA resin to a 2.5 centimeter glass column.

Prepare the resin for protein purification according to the manufacturers and S instructions. Once the samples have been centrifuged, pour the soluble fraction from the ultra centrifugation tubes over the nickel NTA column. Collect the unbound proteins in the flow through fraction.

Continue to wash the column with 100 milliliters of buffer B and collect the flow through. Preserve the flow through fractions at 277 K.Elude the protein in 15 numbered 1.5 milliliter einor tubes. Close the column valve and load one milliliter of buffer C onto the column.

After a three minute incubation time, open the valve and collect the flow through. Repeat this step three times. Continue to elude the protein at one milliliter buffer increments without the three minute incubation.

Using a UV spectrophotometer, locate the protein peak in the fractions by measuring the optical density at 280 nanometers. Finally, perform an SDS page analysis of the collected fractions. The unbound protein flow through the wash, flow through and an ultracentrifugation pellet sample from the gel.

Determine which eluded fractions have high protein levels merge the selected fractions. To remove the 10 hiss tag, add 10 microliters of bovine thrombin per one milligram of protein to the combined sample. Next, dialyze the sample against buffer D to remove the excess in midol and decrease the sodium chloride concentration.

In preparation for ion exchange chromatography, cut the desired length of seven kilodalton molecular weight cutoff dialysis tubing. Wash the tubing with DDW and make a bag by tying a knot At one end, transfer the thrombin digested protein solution into the bag, leaving some space at the top. Clamp the dialysis tubing closed.

Submerge the dialysis tubing into four liters of chilled buffer D.Stir the solution slowly overnight at 277 K after dialyzing overnight, remove the tubing from the beaker. Make a small cut and transfer the protein to a 50 milliliter Falcon tube ion exchange chromatography is used to further purify the memay Delta 41 protein. Connect a pre-packed mono Q4 0.6 100 PE column to an FPLC ACTA purifier system.

Wash the column with five column volumes or CVS of filter DDW equilibrate the column with five cvs of buffer D.Wash the injection loop with two loop volumes of DDW followed by two loop volumes of buffer D.Load the protein into the injection loop. Inject the protein sample at a flow rate of one milliliter per minute and begin collecting fractions. That will be used for SDS page detection of unbound proteins.

Continue to load and inject the protein until the entire sample is loaded onto the column. Wash the column with three cvs of buffer D.To remove any unbound proteins, elude the protein over 60 minutes at one milliliter per minute using a linear gradient of sodium chloride starting at 40 millimolar and ending at two molar. Collect the protein peaks as they appear in the 280 nanometer chromatogram after 100%buffer E has been reached.

Clean the prepec column according to manufacturers recommendation. Perform SDS page analysis of the collected FPLC fractions including the unbound protein and protein peak flow through using prestained markers. Evaluate the protein bands to identify the fractions containing protein at the predicted molecular weight of Mame.

Delta 41 merge the selected fractions in preparation for size exclusion chromatography. The sodium chloride concentration of the merged samples is lowered by dialyzing as previously described against buffer f. After overnight dialysis.

Use a tabletop centrifuge to concentrate the protein sample using a centrifugal concentrator with a 10, 000 molecular weight cutoff. Concentrate the protein to eight milligrams per milliliter at 4, 000 RPM. Validate the desired concentration by measuring the OD at 280 nanometers in a quartz vete.

Assemble the high load 26 60 superdeck 200 size exclusion pre-packed column on an FPLC ACTO purifier system. Wash the column with three cvs of filtered water and equilibrate the column with three cvs of buffer F.Wash the injection loop with two loop volumes of DDW followed by the same volume of buffer F.Once the FPLC has been washed and equilibrated, load the protein sample into the injection loop. Inject the protein sample and start collecting protein peak fractions as they appear in the 280 nanometer chromatogram.

Let the buffer flow until the column void volume is reached. Clean the pre-packed column according to the manufacturer's recommendation. Perform SDS page analysis of the collected fractions and evaluate the gel.

The protein should appear at the correct predicted molecular weight according to the protein pre-sent marker and only one band should be visible. Merge the pure protein fractions and concentrate the protein sample to greater than 20 milligrams per milliliter using a centrifugal concentrator with a 10, 000 molecular weight cutoff and a tabletop centrifuge. Validate the desired concentration by measuring the OD at 280 nanometers.

Confirm the MAME delta 41 sample purity and identity using matrix assisted laser desorption ionization formal D tof. Finally divide the concentrated purified MAME delta 41 protein into pre-marked eend orph tubes at a volume of 25 to 50 microliters each. The sample can be flash frozen in liquid nitrogen and stored at 193 K.This protocol should result in a highly pure size homogenous concentrated MAME Delta 41 protein sample SDS page analysis of the nickel NTA elution profile reveals high levels of memay delta 41 10 hiss protein in the elution fractions at approximately 22 kilodaltons.

The SDS page analysis will also reveal any memay delta 41 protein in the unbound protein flow through the wash, flow through and the ultracentrifugation pellet sample. If large bands around 22 kilodaltons do appear in these samples, troubleshooting is necessary. Ion exchange chromatography is performed to separate the MAME Delta 41 protein from other proteins in the sample through ion exchange.

Under increasing sodium chloride concentrations, the ion exchange chromatogram reveals good separation between two populations of proteins under increasing sodium chloride. Concentration analysis of the samples by SDS page reveals the molecular weight of each population. The first peak is MAME delta 41, while the second peak is the MAME delta 41 10 hiss protein that was not cleaved by bovine thrombin.

If ion exchange chromatography does not result in fully resolved protein peaks, troubleshoot buffer preparation and the slope of the sodium chloride gradient. The final step of the MAME delta 41 purification is size exclusion chromatography. The chromatogram reveals a single peak eluding at 20 kilodaltons.

The appropriate monomer size of the MAME Delta 41 protein maldi to is done to verify the purified MAME delta 41 protein. This analysis reveals two species, one representing the ly charged protein at 20, 347 Daltons and the other representing the doubly charged protein at 10, 176 Daltons. The predicted molecular weight for the recombinant mam MA delta 41 protein following hist tag removal is 20, 597 Daltons.

The small 250 Dalton difference may be due to the common degradation of the first methionine in the second glycine. The results reveal highly purified MAME delta 41 protein. It can be used for further experiments.

We've just shown you how to purify a recombinant syllable protein When performing this procedure. It's important to remember to adjust the buffers to your unique proteins and to perform these experiments as fast as possible. So that's it.

Thanks for watching and good luck with your experiments.