Protein purification

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1. Purification of Recombinant Proteins Sumedha Bobade Ph.D Scholar Animal Biotechnology 2. Recombinant Protein Purification ã Immobilized metal affinity chromatography…
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  • 1. Purification of Recombinant Proteins Sumedha Bobade Ph.D Scholar Animal Biotechnology
  • 2. Recombinant Protein Purification • Immobilized metal affinity chromatography (IMAC) It is widely used method for purifying proteins of interest based on well developed recombinant DNA and protein expression technologies. All proteins are purifies using the method regardless of solubility of the proteins produced in expression host cells. • When the recombinant protein fused to peptide or protein tag such as polyhistidine (His), glutathione –S-transferase (GST) ,Maltose binding protein (MBP) or Step –tag II the properties of tag can be exploided for purification purpose.
  • 3. Steps to produce recombinant protein • 1.Amplification of gene of interest. • 2.Insert into cloning vector. • 3.Sub cloning into expression vector. • 4.Transformation into protein expressing bacteria (E coli) or yeast. • 5.Test for identification of recombinant protein. • 6.Large scale production. • 7.purification.
  • 4. Expression and sample preparation • Components of the expression system • A protein expression system includes, among other things, a vector with an appropriate promoter and other regulatory sequences, along with the gene encoding the recombinant protein of interest. Vectors are available commercially for the expression of recombinant proteins either fused to a tag or untagged. Such expression vectors are designed with control regions to suit the specific host (for example, E. coli versus mammalian cells) and type of expression needed. • Choice of host • The choice of host affects not only the expression of the protein but also the way in which the product can be subsequently purified. In order to decide which host is most suitable, the amount and the degree of purity of the product, as well as its biological integrity and potential toxicity, should be considered. For example, bacterial expression systems are not suitable if posttranslational modification is required to produce a fully functional recombinant product. The location of product within the host will affect the choice of methods for isolation and purification of the product.
  • 5. Expression and sample preparation • Strain of E. coli for Protein Expression : • For high-level protein production purposes, BL21(DE3) is an appropriate E. coli strain. It has the advantage of being deficient in both lon and ompT proteases and it is compatible with the T7 lacO promoter system. For eukaryotic proteins, it is often important to use BL21(DE3) derivatives carrying additional tRNAs to overcome the effects of codon bias. Historically, ampicillin has been the most commonly used antibiotic-selection marker, but itis being replaced by carbenicillin, which is more stable. Vectors encoding resistance to kanamycin or chloramphenicol are now widely used as well. • The difference between BL21 and BL21(DE3) competent E.coli cells? • Both strains are B strains and thus both are deficient in Lon protease (cytoplasm) and OmpT protease (outer membrane). Accordingly, B strains are generally preferred for recombinant protein expression. The DE3 designation means that respective strains contain the λDE3 lysogen that carries the gene for T7 RNA polymerase under control of the lacUV5 promoter. IPTG is required to maximally induce expression of the T7 RNA polymerase in order to express recombinant genes cloned downstream of a T7 promoter.
  • 6. Choice of vector • The choice of vector family is largely governed by the host. Once the host has been selected, many different vectors are available for consideration, from simple expression vectors to those that contain specialized sequences needed to secrete the recombinant proteins. In order to clone the gene of interest, all engineered vectors have a selection of unique restriction sites downstream of a transcription promoter sequence. Recent developments in cloning technology provide increased flexibility in the choice of host and vector systems, including options allowing the DNA sequence of interest to be inserted into multiple types of expression vectors. • The expression of a recombinant protein fused to a tag of known size and biological function can greatly simplify subsequent purification and detection (for expression method development and purification).
  • 7. Choice of tag • There are several affinity tags that can be used to simplify protein purification. The choice of tag may depend on many different factors. The most common tag, the histidine tag, is often a (histidine)6, but other polyhistidine tags consisting of between four and 10 histidine residues have been used. The latter provides for the strongest affinity for the chromatography medium. Other important tags are the GST and MBP tags, both of which are proteins, and Strep-tag II, which is a peptide optimized for chromatography on Strep-Tactin™ based chromatography media. • The rationale for the choice of an N-terminal hexahistidine is manifold. First, an N-terminal tag ensures that the bacterial transcription and translation machineries always encounter 5′ and N-terminal sequences that are compatible with robust RNA synthesis and protein expression, respectively. Second, oligohistidine-tagged proteins can be purified using a relatively simple protocol using immobilized metal affinity chromatography (IMAC) .
  • 8. The histidine tag • The DNA sequence specifying a string of six to nine histidine residues is frequently used in vectors for production of recombinant proteins. The result is expression of a recombinant protein with a 6xHis or poly-His-tag fused to its N- or C-terminus. • Expressed His-tagged proteins can be purified and detected easily because the string of histidine residues binds to several types of immobilized metal ions, including nickel, cobalt and copper, under specific buffer conditions. In addition, anti-His-tag antibodies are commercially available for use in assay methods involving His-tagged proteins. In either case, the tag provides a means of specifically purifying or detecting the recombinant protein without a protein- specific antibody or probe. • Protein purification • As a chromatographic procedure, IMAC has the advantages of having strong, specific binding, mild elution conditions and the ability to control selectivity by including low concentrations of imidazole in chromatography buffers. There is a broad array of common resins with slightly different binding capacities and binding strengths, but all tolerate harsh cleaning procedures.
  • 9. Various purification methods • Affinity chromatography • Based on Charge: Isoelectric Focusing • Based on Hydrophobicity: Hydrophobic Interaction Chromatography • Based on Size: size exclusion chromatography • Immunoaffinity purification • Fast Protein Liquid Chromatography (FPLC) Methods • Reverse Phase HPLC (RP-HPLC)
  • 10. Affinity chromatography using His-tag • In this technique adding extra amino acid to the protein that give it a new property which is bind very tightly to a particular substance.This binding then can be use to isolate the protein away from other material in the cell which do not bind this substance.This method is called as Affinity chromatography using His-tag to purify protein. • A his tag is a a string of (usually six) histidine residue when the gene is cloned . • Commercial plasmid vectors are available that already contain the codon for this his-tag ,making the production of such protein relatively straightforward. • The presence of extra his-tag on the protein often does not interfere with the ability of the protein to fold correctly and carry out its normal function ,but it does make the protein bind very tightly to metal ion such as Ni2+ .
  • 11. Affinity chromatography • A commercial resin is available that has Ni2+ ions attached to it ,so if this resin is placed in a column and a crude extract of E.coli cells that contain the His-tagged protein is passed down the column, the E. coli will pass through the column but the protein with attached his tag will bound to the column • If a buffer containing a salt such as imidazole is subsequently passed down the column, the bound his-tagged protein is now released from the nickel ions, and can be collected in the buffer as it flows out of the column. •
  • 12. Immobilized metal affinity chromatography (IMAC) • beaded agarose or magnetic particles can be derivatized with chelating groups to immobilize the desired metal ions, which then function as ligands for binding and purification of biomolecules of interest. This basis for affinity purification is known as immobilized metal affinity chromatography (IMAC). IMAC is a widely-used method for rapidly purifying polyhistidine affinity-tagged proteins, resulting in 100-fold enrichments in a single purification step. • The chelators most commonly used as ligands for IMAC are nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA). Once IDA- agarose or NTA-agarose resin is prepared, it can be "loaded" with the desired divalent metal (e.g., Ni, Co, Cu, and Fe). Using nickel as the example metal, the resulting affinity support is usually called Ni- chelate, Ni-IDA or Ni-NTA resin. The particular metal and chelation chemistry of a support determine its binding properties and suitability for specific applications of IMAC. • Affinity purification of His-tagged fusion proteins is the most common application for metal-chelate supports in protein biology research. Nickel or cobalt metals immobilized by NTA-chelation chemistry are
  • 13. Advantages and disadvantages for using tags in fusion proteins • Advantages • 1) Simple purification is possible using Affinity Chromatography . Improve protein yield • (2) Detection of the tag instead of the target protein moiety allows for a generic detection method in, e.g., protein production platforms for structural biology. Prevent proteolysis • (3) Facilitate protein refolding : Some tags allow strong binding to chromatography media in the presence of denaturants, making on column refolding possible. • (4) Protect the antigenicity of the fusion protein • (5) Increase solubility: Solubility and stability can be improved. • (6) Increased sensitivity. • 7) Targeting information can be incorporated into a tag. • 8) A marker for expression is provided.
  • 14. Advantages and Disadvantages • Disadvantages • (1) A change in protein conformation • (2) Lower protein yields • (3) Inhibition of enzyme activity • (4) Alteration in biological activity • (5) Undesired flexibility in structural studies • (6) cleavage/removing the fusion partner requires expensive protease • (7) toxicity. • 8) Tag may interfere with protein structure and affect folding and biological activity. • 9) If tag needs to be removed, cleavage may not always be achieved at 100%, and sometimes amino acids may be left. • Untagged proteins • Advantage: Tag removal is not necessary. • Disadvantages • Purification and detection not as simple. • Problems with solubility and stability may be difficult to overcome, reducing potential yield.
  • 15. Ion Exchange Chromatography • It is performed in a pH gradient in an electric field. • The charged proteins migrates towards the anode or the cathode according to the sign of their net charge until they reach the position in the pH gradient where there net charges are zero. • This pH value is isoelectric point (PI) of the substance,an exactly defined physiochemical constant. • Since the molecule is no longer charged it stays there the electric field does not have any influence on it. The protein diffuse away it will gain the net charge and applied electric field will cause it to migrate back to its pI .this concentrating effect leads to the name focusing and makes the method very useful for purification purpose
  • 16. Ion Exchange Chromatography
  • 17. Size Exclusion Chromatography: Gel filtration chromatography - separation by size• It is also known as gel filtration chromatography is a technique for separating proteins and other biological macromolecules on the basis of molecular size . • The solid phase matrix consist of porous beads (100-250 μm) that are packed into a column with a mobile liquid phase flowing through the column. The mobile phase has access to both the volume inside the pores and the volume external beads . The high porosity typically leads to a total liquid volume of ˃ 95% of the packed column. • Separation can be visualized as reversible portioning into the two liquid volumes. Large molecules remain in the volume external to the bead since they are unable to enter the pores.
  • 18. Size Exclusion Chromatography: Gel filtration chromatography - separation by size
  • 19. Chromatography on Hydroxyapatite: Hydrophobic interaction chromatography (HIC) • Hydrophobic interaction chromatography (HIC) separates molecules based on their hydrophobicity. HIC is a useful separation technique for purifying proteins while maintaining biological activity due to the use of conditions and matrices that operate under less denaturing conditions. • 2.The principle for protein adsorption to HIC media is complementary to ion exchange and size exclusion chromatography. Sample molecules containing hydrophobic and hydrophilic regions are applied to an HIC column in a high-salt buffer. The salt in the buffer reduces the solvation of sample solutes. As solvation decreases, hydrophobic regions that become exposed are adsorbed by the media. The more hydrophobic the molecule, the less salt is needed to promote binding. Usually a decreasing salt gradient is used to elute samples from the column in order of increasing hydrophobicity. Sample elution may also be assisted by the addition of mild organic modifiers or detergents to the elution buffer.
  • 20. Chromatography on Hydroxyapatite: Hydrophobic interaction chromatography (HIC) • 3.The method of hydrophobic interaction chromatography (HIC) is based on the observation that protein molecules can interact with fully hydrophobic adsorbents, and this interaction is dependent on the salt concentration of the solution. Similarly to the salting-out of proteins where the increasing salt concentration will lead to the aggregation and precipitation of protein molecules via the rearrangement of their hydrate shell, during HIC chromatography a high salt concentration (1-1.5 M neutral salt) facilitates the interaction between the hydrophobic chromatographic medium and the hydrophobic patches present on protein molecules. During separation, the decrease in salt concentration will lead to the elution of bound molecules. • 4.Hydroxyapatite (HT/HTP) is a crystalline form of calcium phosphate with the molecular formula Ca10(PO4)6(OH)2 . When the HT/HTP is equilibrated with phosphate buffer it is then appears that positively charged proteins interacts nonspecifically with the general negative charged on the column produced by immobilized phosphate ions.
  • 21. Chromatography on Hydroxyapatite: Hydrophobic interaction chromatography (HIC) • 5.The protein then can be eluted by increasing the phosphate concentration ,addition of a salt such as sodium chloride or by the use of Ca2+ or Mg 2+ , which complex with phosphate ions on • Hydroxyapatite (HT/HTP) has achieved only on limited popularity as a chromatographic material for the purification of proteins. • 6.This is for a variety of reason including difficulties in predicting it . A chromatographic behavior ,is relatively low capacity and the fact that its handling properties are not ideal.
  • 22. Affinity Precipitation Method • Affinity chromatography is powerful protein purification technique ,that exploits the specific interaction between a biological ligand ( eg. Substrate , coenzyme, hormone, antibody or nucleic acid) or its synthetic analog and its complementary binding site on protein. • One of the variation of this technique is Affinity precipitation. • There are two main approaches t oaffinity precipitation .The first is called the bis-ligand or homobifunctional ligand approach .The ligand is bifunctional bearing two identical ligand connected by a spacer arm.If the spacer is long enough each ligand can bind to ligand binding site on a different protein molecule .Oligomeric proteins can bind two or more bis- ligand with the consequent formation of crosslinked lattices . • The second approach to affinity precipitation differs from this in that athe affinity ligand has two function one of which bind the target protein and second to promote the precipitation of the aggregate.
  • 23. Immunoaffinity purification • The immunoaffinity chromatographic purification technique of fusion protein with reference to human interleukin is described • Interleukin 2 is joined to a small DNA sequence encoding a marker peptide (That synthesized 8 amino acid ,Asp-Tyr-Lys-Asp-Asp-Asp- Asp-Lys) produces a fusion protein in yeast (S.cerevisiae) . • The marker peptide has a dual reduces the degradation of interleukin 2 beside helping in its purification.(The eight amoino acid marker peptide is commercially available under the brand name Flag peptide). • The fusion protein interleukin 2 joined to a marker peptide can be purified by immunoaffinity chromatography. • The specific monoclonal antibodies (MAb) against the marker peptide are immobilized on polypropele support. These MAb serves as the ligand with marker peptide antibodies and selectively binds to fusion proteins tagged with marker peptide. • However the remaining protein pass through the immunoaffinity column. The immunopurified fusion protein can be elute later from the column.
  • 24. Immunoaffinity purification
  • 25. Fast Protein Liquid Chromatography (FPLC) Methods • High performance liquid chromatography (HPCL) procedures exploits column packaging with average diameter of as 5-40 μ However these are used in high pressure system often with organic solvent and are generally limited to rather low sample loading. • FPLC provides a full range chromatography mode such as ion exchange, chromatofocusing, gel filtration, hydrophobic interaction and reverse phase based on particles with average diameter sizes in the same range as those used for HPLC saperation.
  • 26. Reverse Phase HPLC (RP-HPLC) • This is well established technique for isolation ,analysis and structural elucidation of peptides and proteins. It is used in protein isolation and purification and have reached peak owing to recent development in high affinity ion exchange and hydrophobic interaction support which are now capable of equivalent levels of resolution to RP-HPLC • The solvent gradient is generally superior to isocratic elution between saperation is then achievable within reasonable time frame and peak broadening of later elution peaks is reduced and thus sensitiveity increased .In gradient elution RP-HPLC, proteins are retained essentially according to their hydrophobic character, the retaintion mechanis can be considered either as adsorption of the solute at the hydrophobic stationary surface or as a partition between the mobile and stationary phase.
  • 27. Manual purification techniques • For small-scale purification of tagged proteins, a single affinity chromatography step with a simple elution by a step gradient is usually sufficient. Manual purification can be performed in batch or by using gravity-flow or spin columns, or 96-well plates. • When a tagged protein is purified by a batch method, the protein sample is added to a purification medium usually in a disposable plastic tube. The chromatography medium is then washed and the tagged protein is eluted. The batch method is suited to purification on a small scale. • A tagged protein can also be purified by simply passing the protein sample through a disposable • column prepacked with an appropriate medium. There are columns esp
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