The overall goal of the following experiment is to produce very pure recombinant proteins by a two-step affinity purification procedure. To achieve this, a bispecific purification tag based on an albumin binding domain is cloned into an expression vector containing the gene of interest and is followed by expression of the fusion protein in e coli. As a second step bacterial lysate containing the target protein is subjected to purification on a chromatographic column with immobilized human serum albumin or HSA.
This is followed by an additional purification on a MAB select superior resistance or sure column containing immobilized Z domain derived from staphylococcal protein A.This results in a highly pure product suitable for demanding applications. Next, the purity is evaluated by SDS page and if necessary, mass spectrometry in order to ensure that the protein of interest was successfully purified to homogeneity, the results show efficient purification after two successive steps as compared to a single step with the novel bispecific purification tag utilized in this protocol. The main advantage of this technique over existing methods like one step affinity purification, is that pure protein can be achieved by a simple protocol utilizing only one affinity handle regardless of the properties or the target protein.
This method can be used for protein with very diverse characteristics. Also, since the tag is very small, it's easily produced by the bacterial host. Generally individuals new to this purification procedure will find it very simple and straightforward.
A critical step is to adjust the pH of your sample before loading it onto the second column. A small purification tag with two inherent affinities was developed by incorporating an additional binding site into an existing albumin binding domain derived from streptococcal protein G.The 46 amino acid albumin binding domain folds into a stable three helix bundle and contains a binding site for human serum albumin, primarily located on the second helix by genetically randomizing 11 surface exposed amino acids located in the first and third helix novel binding surfaces were engineered several rounds of biop panning against a diametric zoma derived from staphylococcal protein A with the combinatorial library expressed on phage, resulted in a bispecific molecule termed A BDZ one with affinity for both HSA and the Z domain. To prepare the bispecific fusion tag expression plasmid prepare the PCR fragments of the A BDZ one gene flanked by suitable restriction sites for ation and terminally of the target gene in the expression plasmid cleave, the A BDZ one PCR fragments and the purified expression vector containing the gene of interest With the chosen restriction enzymes in a suitable reaction buffer purify the products before ligation.
Next ligate the restricted A BDZ one fragment into the expression vector containing the gene of interest and transform the ligation product into e coli. Spread the transformed cells onto agri plates, supplemented with suitable antibiotics for selection. PCR screen a few colonies and sequence verify the resulting expression cassette by DNA sequencing.
Finally, prepare the plasmid from an overnight culture of a sequence verified colony and transformed to the preferred expression strain to express the target protein. With the novel tag fused to its end Termini first inoculate a single bacterial colony into 10 milliliters of tryptychs soy broth supplemented with five grams per liter of yeast extract and the appropriate antibiotics incubate at 150 RPM and 37 degrees Celsius overnight. The following day, inoculate one milliliter of overnight culture in 100 milliliters of fresh, medium, and induce protein expression.
When the cells reach the logarithmic growth phase. After protein induction incubate the cells at 150 RPM and 25 degrees Celsius overnight, harvest the cells the next day by centrifugation orthogonal affinity purification of the A BDZ one fusion protein is achieved through initial purification on either a column with immobilized HSA or zoma. A successive purification step is then performed, employing the second affinity of the bispecific tag.
Chromatographic media with the Z domain immobilized such as MA select sure are readily available for purification of antibodies. Whereas HSA can be chemically coupled to media to provide the second matrix to proceed with orthogonal affinity purification Resus, suspend the pellet in 25 milliliters of running buffer. Then disrupt the cells by sonication at 60%amplitude and pulses of one second, followed by a one second pause for a total of three minutes.
After centrifuging the sample, filter the target protein containing super natin through a 0.45 micron filter before further purification. Next equilibrate a one milliliter HSA spheros column or an NHS activated column coupled with HSA with 10 column volumes of running buffer at one milliliter per minute on a suitable protein purification system. Once equilibrated load the bacterial lysate at 0.5 milliliters per minute, reset the flow rate to one milliliter per minute and wash the column with five column volumes of running buffer, followed by five column volumes of washing buffer.
Elute the sample with elution buffer at one milliliter per minute and collect the fractions. Monitor absorption at 280 nanometers to select fractions from the Eluded peak. For further purification, pool the fractions with the highest protein concentration, then dilute two times.
Ensure running buffer and ensure the pH is around neutral. This step is critical for protein binding to the Z domain. Add one molar tris HCL pH eight to increase pH if necessary.
Next, load the sample at 0.5 milliliters per minute onto a one milliliter high trap MA, select sure column that has been equilibrated with sure. Running buffer after the flow rate has been reset to one milliliter per minute, the column is washed with five column volumes of sure running buffer. Then elute the proteins with 0.2 molar acetic acid pH 2.7 for sensitive target proteins.
The EIT can be neutralized directly upon collection by addition of T tris. H-C-L-S-D-S page can be used to evaluate protein purity. It is important to fully reduce the sample since a free cyte in A BDZ one may cause dimerization of the product under non reducing conditions.
As a final step, the molecular weight of the purified product can be analyzed by mass spectrometry following purification by the orthogonal protocol on an HSA column, followed by a MA select sure column. Different samples collected at several purification points were analyzed by SDS page. The samples included the A BDZ one tag itself, as well as three different human target proteins fused to AB DZ one representing different solubility classes, molecular weights, and isoelectric points.
Lanes one through four show protein lysates before purification for the three different AB DZ one fusion proteins and the AB DZ one tag itself. Lanes five through eight show the results from the HSA purification step of the proteins. Finally, lanes nine through 12 result from the final mAbs select Sure purification step.
In addition, samples acquired from the same lysates purified in the reverse order on the same columns were analyzed here. Lanes one through three show lysates before purification, whereas lanes four through six were taken after the first purification step on the MAB Select Sure column. The high purity products in lane seven through nine result from the final HSA purification.
The results clearly show the utility of a dual tag and two highly specific purification steps. Even though reasonable purity is achieved after the initial purification step as seen from the gels, the successive step yields a very pure product well suited for highly demanding applications. Once mastered, the purification step central to this method can be done in hours by one person to achieve a highly pure recombinant protein suitable for many different applications.
For example, following this procedure, the purified biomolecules can be used for other applications, such as interaction studies in order to answer questions like the binding strength of a complex. After watching this video, you should have a really good understanding of how to achieve a real pure protein product by utilizing this dual affinity tag.
