Affinity chromatography

Affinity chromatography

1. AFFINITY CHROMATOGRAPHY

1.1. INTRODUCTION

In affinity chromatography biomolecules are separated according to the ligand specificity property, biological function and chemical structure. Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins)

and a specific ligand coupled to a chromatography matrix. The technique offers high selectivity,high resolution. This technique is ideal to capture an intermediate step in a

protocol. The biochemical interactions can be of various types like the electrostatic

forces, hydrophobic interactions, vanderwaals force or even hydrogen bonds.

1.2. COMMON STEPS IN AFFINITY CHROMATOGRAPHY

Purification

  1. Affinity media is equilibrated with binding buffer.
  2. Sample is applied such that the specific binding of the

target molecule to a complementary binding substance

(the ligand) is favoured . Unbound material washes through

the column.

  1. Target protein is recovered from the ligands by

changing the conditions to favour elution

Elution can be both specific and non-specific.

Specific elution is performed using competitive ligand

and non-specific by changing pH, ionic strength or polarity.

  1. Affinity medium is re-equilibrated using binding buffer.

1.3. SELECTION OF MEDIA

Specific affinity media are prepared by coupling a ligand to a selected gel matrix,

following recommended coupling procedures.

GE Healthcare sell pre packed Hi Trap Affinity columns for method optimisation or small scale purification. The selection guide for these affinity matrices is attached in the appendix. (APPENDIX )

1.4. PREPARATION OF MEDIA AND BUFFERS

The solutions that we use in affinity chromatography should be washed thoroughly before using.

If the manufacturer supplies a freeze-dried product it should be re-swelled in a proper buffer. High quality chemicals and waters should be used. Filters of size 0.45 μm or 0.22 μm should be used.

Buffers can be reused only for identical products to avoid cross-contamination. However, the re usability depends upon the sample. If the affinity medium is used regularly care should be taken that the contaminants are removed from the crude product so that the ligand is not damaged. Only mild rotation should be followed. Magnetic stirrers should be avoided because they cause damage to the matrix.

1.5. SAMPLE PREPERATION AND APPLICATION

Sample must be purified before usage to avoid clogging in the column. The interaction between the target and the ligand should not be either too high or too low, to get better yields.

In affinity chromatography sample volumes does not effect the does not affect the separation. The sample can also be applied in aliquots. Composition of sample, pH and flow rate should be adjusted to desirable limits. The column must be pre-equilibrated with the binding buffer. The elution of the target should be started only after all the unbound material is washed out.

1.6. ELUTION METHODS

1.7. FLOW RATES

Flow rates depend on the dissociation rates on ligand/target molecule interaction. Determination of flow rate is necessary to achieve efficient binding, to maximize recovery from elution, to minimize total run times for column re-equilibration. To maximize the benefits low flow rates should be used.

1.8. ANALYSIS OF RESULTS AND FURTHER STEPS

The quality of the eluent shows from the first step is an indication to check if the sample needs to be purified further. Further a second technique (some other applicable chromatographic technique ) can be used to cross check the results. A desalting column can also be used .

1.9. AFFINITY TAGS

Affinity tags are the most effective tools for protein purification. Affinity tags helps to purify proteins without prior knowledge of their biochemical properties. Examples of some commonly available tags are listed in table below.

Comparison of some commonly used tags

Name

Length, AA

Binds to

Eluted with

Comment, including purification costs

CBP, calmodulin binding protein

28

Calmodulin

EGTA

Moderately expensive

FLAG; DYKDDDDKG

8

Anti-FLAG antibody

FLAG peptide

Expensive

GFP; green fluorescent protein (or variations such as EGFP, or similar such as YFP)

~220

-

-

Detected by fluorescence or antibodies

GST, glutathione S-transferase

218

Glutathione

Glutathione

Cheap

HA; influenza virus hem agglutinin; YPYDVPDYA

9

Anti-HA antibody

HA peptide

Expensive

Hexa-histidine, His6

6

Nickel

Imidazole

Cheap

IgG Fc, immunoglobulin Fc domain

220, may vary

Protein A or G

Low pH or other denaturants

Moderately expensive; if the tag includes disulphide bonding sites, the recombinant protein may take a dimeric form

Lac Z; β-galactosidase of E. coli

~1000

-

-

Detected using colorimetric enzyme substrates or antibodies

MBP, maltose binding protein

396

Amy lose

Maltose

Cheap

Myc, c-myc; EQKLISEED

10

Anti-Myc antibody

Myc peptide

Expensive

Protein A

247

Immunoglobulin

Low pH or other denaturants

Cheap; unlike the Fc tag, this tag does not have sites for disulphide bond formation; however, unlike protein A columns, immunoglobulin columns are more fragile

STR, streptavidin II

8

Streptactin

Desthiobiotin

Expensive

Thioredoxin; E. coli

109

Thiobond resin

-

Expensive

V5; of P/V proteins of SV5 virus; GKPIPNPLLGLDST

14

Anti-V5 antibody

V5 peptide

Expensive

VSV; from vesicular stomatitis virus; YTDIEMNRLGK

11

Anti-VSV antibody

VSV peptide

Expensive

Affinity chromatography is simple and alternative method for the purification of recombinant proteins. It utilises the utilises a fusion of short amino acid sequence as a tag to the recombinant proteins. Examples of techniques exploiting this strategy are :

  • The glutathione- S- transferase system (GST) fusion system used together with glutathione -Sepharose beads.
  • Protein A fusion system combined with immunoglobulin columns.
  • Epitope- tagging used with specific antibodies.
  • The histidine tagging system for metal chelate affinity chromatography.

2. GST- TAGGED PROTEINS

2.1. THE GST TAG

The GST tag is a 211 AA protein whose DNA sequence is usually integrated into the host vectors for the purification of recombinant proteins. As a result a GST fusion protein is obtained in which the in which the functional GST protein(26 kDa) is fused to the N-terminus of the recombinant protein. The tag provides a means by which the recombinant protein can be purified or detected without a protein specific antibody or probe.

2.2. MECHANISM OF ACTION OF GST PROTEIN PURIFICATION

Glutathione S- transferase has a specific substrate Glutathione which is tripeptide. Pure GST or GST tagged proteins are captured to the solid support by enzyme- substrate binding reaction. The reduced Glutathione is immobilized through its sulfhydryl group to solid support such as cross-linked beaded agarose. Binding is effective in neutral (physiological) buffers such as Tris- buffered saline with pH 7.5. Binding should retain the essential enzymatic action , structure but should not denature the protein. The affinity column should be washed off to remove the non-bound samples. The purified GST protein can be dissociated and eluted by adding excess of free reduced glutathione. The free glutathione competitively displaces the immobilized glutathione binding interaction with the GST, allowing the fusion protein to move out of the column.

2.3. MATERIALS/SOLUTIONS THAT CAN BE USED FOR GST FUSION PROTEINS

Lysis buffer

(decreases non-specific binding)

will vary, but 0.25 M NaCl can be added before loading the column.

Equilibration Buffer

(for equilibrating the column.)

20 mM Tris, pH 7.5

0.25 M NaCl

2 mM EDTA

2 mM EGTA

1% Triton X-100

0.03% Brij-35

Wash Buffer

20 mM Tris, pH 7.5

0.25 M NaCl

2 mM EDTA

2 mM EGTA

0.03% Brij-35

no Triton!!

Elution Buffer

Wash Buffer + 20 mM glutathione, pH 8.0

(61 mg glutathione/10 ml buffer; add 13 ul 10M NaOH

Dialysis Buffer (will vary)

50 mM Tris, pH 7.5

0.1 mM EDTA

0.1% 2-mercaptoethanol

50% glycerol

2.4. GENERAL PROCEDURE (adapted from Amersham's)

  1. Prepare glutathione-sepharose beads.
  2. Lyse cells
  3. Bind proteins to (by mixing and incubation of lysed cells mixed with sepharose beads.)
  4. Transfer to column , then continue with draining the column instead of pelleting beads and pouring off supernatants.
  5. Wash unbound protein from beads.
  6. Elute protein from beads.
  7. To monitor the fractions use Bradford assay.

2.5. ADVANTAGES OF GST TAG OVER THE OTHER TAGS

  • Appears to function as a discrete domain
  • It can be detected immunologically with sera
  • Relatively small in size
  • Low immunogenecity makes it ideal for raising antisera to fusion proteins.

2.6. LIMITATIONS

  • The functional GST protein (26kDa) is larger than many other fusion protein affinity tags.
  • The structure of the GST tagged protein often degrades upon denaturation and reduction for protein Gel-electrophoresis. As a result, electrophoresed samples often appear as a ladder of lower MW bands below the full-sized fusion protein.
  • Glutathione interferes with Lowry and BCA assays, but Bradford works fine.

3. EXAMPLE 1

EXPRESSION AND PURIFICATION OF GLUTATHIONE S-TRANSFERASE TAGGED HIV-1 gp 120 : NO EVIDENCE OF AN INTERACTION WITH CD26

3.1. BACKGROUND

A variety of recombinant forms of gp120 have been reported and the native folded molecule is distinct immunogen . So there has been a increased interest in the purification of molecule with minimum denaturation. The secreted protein can be purified with minimum denaturation on immobilised glutathione and it is active as the parentral molecule in dinding to CD4. So GST tag is used for the expression and purification of surface glycoprotein HIV 1 (gap 120) fused to carboxy terminus by the use of new bacillovirus expression system

In this paper there is a description of a new form of gp120, GST -gp 120 specially designed to enable the identification of ligands of the molecule.

3.2. RESULTS AND DISCUSSION

BACULOVIRUS VECTOR , IMPROVEMENTS AND CONSTRUCTION

  • Baculovirus allows the expression of GST fusion vectors in insect cells for subsequent purification using a affinity matrix.
  • Normally the Glycosylated proteins are secreted to the cell surface or external milieu. So for affinity purification of such proteins further more improved baculovirus transfer vectors pASG2T - tag were constructed. In these improved vectors the GST coding region is appended in phase to a signal peptide sequence derived from the baculovirus major surface glycoprotein gp67.
  • In this new vector the cloning sites are retained in the 3' end and an additional monoclonal epitope tage is added to 3' to the poly linker cloning sites.
  • The preliminary experiments from the pASG2T - tag to generate recombinant baculovirus showed that GST could be efficiently secreted from insect cells and purified from tissue culture medium by chromatography on glutathione agarose.

GENERATION OF gp-120 FUSION PROTEIN

A gp-120 clone suitable for the fusion to GST reading frame without encoding intervening hydrophobic sequence is generated by amplification of gp-120 domain of HIV-1 LAI. Recombinant baculovirus AcSG2T-120 constructed using the transfer vector was used to infect sf9 cells , and the expression of GST-gp120 was confirmed by SDS PAGE and western blotting. From western blot it is identified that GST-gp120 was secreted efficiently into the culture medium. Purified protein showed a single band ~140kDa on stained gels with no significant denaturation.

YIELDS OF PROTEIN

  • Yields of protein obtained were comparable to those obtained without fusion to GST. Some dimers appeared in the western blots. These dimers are due to the cross linking between monomers during secretion.
  • CD4 binding with the GST-gp120 is comparable to that of purified gp120 obtained by ELISA assay.

TESTING THE ABILITY OF GST-gp 120 BINDING TO CD26

To test this three assay formats were used

CO-PRECIPITATION ASSAY

sf9 cells are co-infected with recombinant miraculous expressing GST-gp120 and either CD4 or CD26. The expressed proteins were purified by affinity chromatography on glutamate arose and analysed by western blot. Co-purification of CD4 occurred due to co-expression of CD4 with GST-gp120. There was no evidence of interaction between GST-gp120 and CD26 during co-expression.

ELISA FORMAT

Purified GST - gp120 was used to directly coat a solid surface and incubated with CD4 or CD26. No evidence of interaction of CD26 with GST - GP 120 was identified both in presence and absence of CD4 with this ELISA format

PROTEIN OVERLAY ASSAY

Blots were prepared using gp120, CD26 and sCD4 and incubated with either GST-gp120 or St only followed by an anti-GST serum and conjugate. Results showed there is a strong binding with CD4 but no binding with CD26. These results further suggest that there is no interaction between CD26 an dgp120.

GST-gp120 may also be a improved agent for structural studies.

3.3. CONCLUSION

Experiment achieved concentrations of GST-gp120 in excess of 20mg/ml but the crystal structures were not determined.

4. EXAMPLE 2

AFFINITY PURIFICATION OF GST FUSION PROTEINS FOR IMMUNOHISTOCHEMICAL STUDIES OF GENE EXPRESSION

4.1. BACKGROUND

Usually researchers rely on the automatic application of one standard purification recipe to any fusion protein. In this paper they they described a method that is simple but general procedure that can be applied for each particular construct to obtain milligrams quantities of GST fusion proteins.

4.2. MATERIALS AND METHODS

PROTEIN SOLUBILIZATION

The expression constructs used in this study were prepared and bacteria is treated with Lysol. The lysozyme treated bacteria were added to ST/LOX6.Sonicated and re suspended in STE buffer. The samples were tested by PAGE and SDS for solubility of fusion proteins.

BINDING TO GA BEADS

Triton X-100 was added at different concentrations to the supernatant. The lowest Triton-X binding was selected and applied to remainder of the supernatant.

ELUTION

The GA beads were treated with variety of buffers to elute the proteins. The remaining proteins were incubated and boiled to elute the proteins that are still bound.

4.3. RESULTS

Challenges!

Answers..

Early purification attempts using standard GST/LOX6 protein was insoluble in the absence of denaturing detergents and was lost at early purification stage to remove cell debris

The buffer solution used to lyse the cell is replaced with Sarkosyl and Triton X-100. This treatment helped the cells not bind to affinity column.

Excessive sonication produced fusion proteins that subsequently did not bind to GA beads, probably due to denaturation of the GST moiety of fusion protein.

The viscosity was reduced so that the GA beads are not inhibited.

  • Fusion proteins bound to sarkosyl alone bind poorly to GA beads whereas the GA binds efficiently to the mixture containing sarkosyl and Triton X-100.
  • It is proved from the results of this paper that the amount of GA beads required to bind to the protein are several times larger than the ones mentioned in the usual protocols.
  • The yields obtained from this protein purification test were sufficient to produce fusion proteins to immunize animals and make affinity chromatographic column

4.4. DISCUSSION

Optimization procedure for the purification of new GST proteins fusion proteins is shown in the below figure.

This protocol quickly tests different Sarkosyl and Triton X-100 concentrations for fusion protein solubilization, different amounts of GA beads for efficient binding, and different elution buffers.

The isolation procedures often failed due to steps like protein solubilization, binding to GA beads and elution. To overcome these problems several simple tests are made, which resulted in milligram quantities of fusion proteins.

Also due to various problems like rapid over expressionn of the protein under strong bacterial promoters control , improper folding or due to absence of post translational modification the eukaryotic proteins areinsoluble. Thee use of mixtures of ionic and non-ionic detergents has been shown to solubilize these proteins without compromising their ability to bind glutathione, a GST ligand . The ionic detergent (Sarkosyl) is thought to help solubilize the protein by partially denaturing it; the subsequent addition of the non-ionic detergent (Triton X-100) putatively sequesters the first detergent into its micelles and allows the renaturation of at least the GST moiety of the fusion protein, thus, enabling the binding to GA beads. Refolding of transcription factors after solubilization with Sarkosyl from inclusion bodies has also been achieved by slowly diluting the detergent by dialysis.

Another factor that influences the yield of purification is the amount of GA beads used to bind GST proteins. Elution profile is also another important factor for obtaining good yields.

4.5. CONCLUSION

The three elution buffers tested in this experiment are compatible with the direct immunization of animals following elution and with the use of the purified protein for the affinity purification of the antibodies produced by the immunization. The antibodies raised against our fusion proteins were able to detect the endogenous, native protein in fixed tissue. The patterns of expression revealed by antibody staining were essentially the same as those previously reported using in situ hybridization or staining with other antibody preparations.

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