Lecture 8: Affinity Chromatography-III
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1 Lecture 8: Affinity Chromatography-III Key words: Chromatography; Affinity chromatography; Protein Purification During this lecture, we shall be studying few more examples of affinity chromatography. The current lecture ends with home assignments.. Glutathione S-transferase for purification of recombinant proteins: For easy purification of recombinant proteins they are generally tagged with GST (Glutathione S- transferase) protein to create fusion proteins (protein sequence attached with GST sequence). The tag has the size of 220 amino acids (roughly 26 kda), which is quite big compared to other tags like the myc- or FLAG-tag. It generally helps the recombinant protein for soluble expression and further purification. Moreover, a thrombin (a protease) recognition sequence is included in between the GST tag and protein sequence. This helps in removal of GST tag by cleavage with thrombin after purification of fusion protein. The GST part of fusion proteins has affinity for glutathione as glutathione is the substrate for GST. This enzyme (GST) substrate (glutathione) affinity is used for purification of fusion protein. Agarose or other polymer beads can be coated with glutathione, and such glutathioneagarose beads bind GST-proteins. The crude cell lysate can be loaded on a column packed with glutathione-agarose matrix, and washed extensively. The fusion protein will bind with glutathione coated agarose beads through GST, while other proteins will wash off. The elution of bind fusion protein was performed by free glutathione solution. Due to higher concentration of glutathione in solution, fusion protein leaves the glutathione coated beads and comes in solution. The eluted affinity purified fusion protein will now be subjected to thrombin cleavage for removal of GST tag. Amount of thrombin used for cleave is very minute and generally removal of thrombin is not required (or an immobilized thrombin may be used which may be removed by simple centrifugation after completion of cleavage reaction). The thrombin treated protein was again loaded on regenerated column. This time the cleaved GST tag will Page 1 of 5
2 bind with beads and recombinant protein will come in un-bound fraction. A general scheme for purification of proteins with affinity tag is given is the Fig. 1. Maltose-Binding Protein (MBP) for purification of recombinant proteins: In this technique the gene of interest is cloned into pmal vector creating MBP-encoding male gene and factor Xa (a protease) cleavage site. This gene can be expressed in E. coli producing MBP fusion protein (MBP fused with protein of interest containing factor Xa cleavage sequence between MBP and protein of interest). This MBP-fusion protein is purified using amylose column, MBP has affinity for the amylose ligand and finally fusion protein can be eluted using maltose gradient. Finally MBP tag can be cleaved from fusion protein using factor Xa protease (as there is factor Xa cleavage site between MBP and protein of interest). Generally factor Xa protease used for the cleavage is in minute amount and removal is not required (or an immobilized factor Xa protease may be used which may be removed by simple centrifugation after completion of cleavage reaction). Cleaved MBP tag can be separated from the protein of interest by loading it again to amylose column. This time cleaved MBP tag will bind to column but protein of interest will go in unbound fraction. Scheme of an affinity purification method is given in following figure. This could be a very effective method to purify a recombinant protein for which there is no known easy way to purify using substrate-based affinity chromatography (Fig 1) Page 2 of 5
3 1.Clone MBP/6x His/GST encoding sequence 2.Expression MBP/His tag/gst tag Protein of interest sample 3.Purification Washed proteins Recombinant protein with Tag Thrombin/Factor Xa 4.Cleavage 5. Native protein. Figure 1: Purification of recombinant proteins using different affinity tags. Application of Affinity Chromatography in Proteomics Proteomics is the study of total protein pool from cell line, tissue or organism. The most commonly used experimental techniques in proteomics are 2 DE (two dimensional gel electrophoresis) for the separation of proteins from a mixture containing thousands of proteins (one dimension is isoelectric focusing and second dimension is SDS PAGE) and Mass Spectrometry for the identification of separated proteins. In the separation procedure, affinity Page 3 of 5
4 chromatography can also play an important role (Fig. 2). An excellent review article published [Analytical Biochemistry 324 (2004) 1 10] on the topic. MALDI TOF Protein identification by PMF Proteins 2D SDS ESI-MS/MS Protein Mixture mixture MS/MS Proteins mixture LC MS/MS Figure 2: Application of affinity chromatography at different stages of proteomic analysis. Solid circles indicate the position where affinity chromatography can be used. Note: We shall be reading protein identification by Mass Spectra and theory of mass spectrometry in coming classes. Page 4 of 5
5 Home Assignments 1. Explain why protein does not bind to Ni-NTA matrix in acidic ph range. What are different methods of elution of protein from Ni-NTA? 2. A solution containing aspartic acid (PI=2.98), glycine (PI=5.97), leucine (PI=5.98), and lysine (PI=9.74) in a ph 3.0 citrate buffer was applied to a CM-cellulose column equilibrated with the same buffer. The column was then eluted with increasing NaCl concentration. In what order will the amino acids elute from the column? 3. Do a survey of methods to ensure purity of a protein. 4. Read about dye-ligand affinity chromatography Submit your assignment to course developer by ; you will get an reply with grading and feedback in a week time. Page 5 of 5
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