Production of recombinant proteins

Isolation & production of r-proteins

1. Strategies for commercial production , isolation/purification of recombinant protein.

2. What is recombinant protein? Recombinant protein is a manipulated form of protein, which is generated in various ways to produce large quantities of protein modify gene sequence and manufacture useful and commercial products.

3. Production of recombinant protein Transcription and translation steps belong to the recombinant protein expression systems. To make recombinant proteins, the gene is isolated and cloned into an expression vector. Generating a r protein requires the protein expression system, protein purification system, protein identification systems. At DNA level :- 1. finding the protein of interest Sequence has to be determined from wild type protein 2. Deletion mutants:- AA can be deleted from the sequence, either individually or in groups 3. Point mutants:- at any position in the sequence an amino acid can be replaced by another to generate a mutant protein ( this mutant protein is expected to have little or no biological activity ,because histadine 119 is important for that activity )

4. Suitable expression system 1. Bacterial expression system. 2. Yeast expression system. 3. Insect/ Baculovirus expression system. 4. mammalian expression system. Through r DNA technology, a large quantity of proteins can be produced. This involves inserting the desired protein gene into an expression vector which must contain promoter so that the protein can be expressed

5. Stategies Rational designing of proteins by site directed mutagenesis Directed evolution of proteins by random mutagenesis Before engineering characteristics of wild type protein must be determined (variety of analytical methods) Production of mutants protein to assess elements that are necessary for its function

6. Pre conditions Working knowledge of protein Locating a desired protein Clone and express The design and construction of new proteins or enzymes with novel or desired function, through the modification of amino acid sequence

7. There are basically two methods for producing recombinant proteins . One is molecular cloning, a laboratory method used to make recombinant DNA. The other method is the polymerase chain reaction used to proceed the replication of any specific DNA sequence selected. Cloning process :- 1. G ene of interest is cut out with restriction enzymes 2. Host plasmid is cut with same RE 3. Gene is inserted into plasmid and ligated with ligase . 4. New (engineered) plasmid inserted in to bacterium(transformed)

8. Hepatitis B vaccine The gene encoding for Hepatitis B surface antigen ( HBsAg ) has been identified. The HBsAg vaccine as a subunit vaccine, is produced by cloning HBsAg gene in yeast cells

9. Isolation of recombinant protein Recombinant protein are isolated from very complex protein mixture present in producing cell. The isolation process involves in general four mutually interconnected stages:- Release of recombinant protein from the cellular environment. preparation of the specimen for separation Separation. Qualitative and quantitative analysis of the preparation.

10. => in addition to the protein of interest, several thousand other protein with different properties are present in the extract, along with nucleic acid (DNA and RNA), polysaccharide, lipid, and small molecules. the challenge is therefore, is to separate the protein of interest from cell of other components in the cell, especially the unwanted contaminating proteins with reasonable efficiency, speed, yield, and purity while retaining the biological activity and chemical integrity of polypeptide. Extraction process 1. Cell lysis (osmotic shock) -> protein source -> E.coli periplasm intracellular protein 2. Enzymatic digestion -> protein source -> intracellular protein / periplasm 3. Ultrasonication -> cell suspension -> inclusion bodies 4. Manton- gaulin homogeniser -> cell suspension large scale only. Note :- If lysates are to viscous to handle which caused by the presence of high concentration of host nucleic acid give DNAse and RNAse .

11. By exploiting the difference in properties between the protein of interest and other proteins in the mixture, rational series of fractions steps can be designed. These properties include: Size Shape Charge Isoelectric point Charge distribution Hydrophobicity Solubility Density Ligand binding Metal binding Reversible association Post translational modification Specific sequence or structure Biological source : take a tissue/cell type that contain large amounts of the target protein. Lyse cell : separate cell homogenate into fractions

12. Strategies of purification of r-proteins: CAPTURE STEP Isolate, concentrate, and stabilize the target product. Effectiveness assessed based on chromatography speed POLISHING STEP Includes a capture step and one or more polishing steps. Flow rates and load volumes of the polishing steps may be decreased in order to improve resolution. Removes most of the bulk impurities (other proteins, nucleic acids, endotoxins, viruses) INTERMEDIATE STEP

13. Performance parameters Resolution Capacity Speed recovery Increasing the number of purification steps will often decrease the overall protein recovery

14. CAPTURE STEP Definition: Initial purification of the target molecule from the source material. Goal: Rapid isolation, stabilization, and concentration. Removal of critical contaminants to obtain high level of purification. IEX or AC Target protein isolated from critical impurities (proteases and glycosidases ). Maximizing capacity and/or speed will be at the expense of some resolution. Gradient elution to step elution

15. Intermediate step Definition: Further removal of bulk contaminants. Goal: Purification and concentration. Eradication of Impurities like other proteins, nucleic acids, endotoxins, and viruses. The requirements for resolution will depend on the properties of the sample produced from the capture step and the purity requirements for the final product Elution by a continuous gradient

16. Polishing step Definition: Final removal of trace contaminants and adjustment of pH, salts, or additives for use or storage. Goal: End product of required high-level purity. Trace impurities removed (e.g., endotoxins, nucleic acids, or viruses), and closely related proteins such as microheterogeneous structural variants. To achieve enough resolution it may be necessary to sacrifice sample load (overload may decrease purity) and recovery by narrow peak fractionation. Recovery of product in buffer conditions. GF ---- IEX

17. Selection and combination of purification methods

18. AC: Single-step purification of affinity-tagged proteins using AC may give sufficient purity for some applications. AC-GF: Complementing an AC step with polishing by GF is very common, and can often be used for generic setups when regularly purifying multiple proteins. AC-IEX-GF: For high-purity requirements in affinity-tagged protein purification, AC-IEX-GF is a powerful and convenient combination. Untagged proteins can usually be sufficiently purified by combining purification methods that separate on the basis of different physicochemical characteristics of the proteins (orthogonal methods). IEX-HIC-GF: The combination IEX-HIC-GF is a very often used three-step purification because the high-salt conditions after the first step can simply be adjusted with additional salt for HIC purification and followed by GF for polishing and salt removal. HIC-IEX-GF: If ammonium sulfate precipitation has been performed, the combination HIC-IEX-GF is suitable because HIC requires high-salt conditions for binding and gives elution in a relatively low salt concentration in a significantly smaller volume. Dilution or desalting can then be used to remove remaining salt, so that the sample can be bound to an IEX column.

19. Purification of untagged r-protein by column chromatography: Various chromatographic techniques developed based on protein molecules size, shape, overall charge, presence of surface hydrophobic groups and ability to bind various ligands. Chromatographic Techniques commonly used and their basis of separation are as follows: Ion exchange – protein surface charge at given pH Hydrophobic Interaction Chromatography – surface hydrophobicity of proteins Gel filtration chromatography – mass or size/shape of proteins Chromatofocussing – isoelectric points Affinity chromatography – biospecific interaction between protein and a ligand

20. Chr media The particle size of the medium strongly affects efficiency and flow resistance. In the polishing stage, focus is put on high purity, which can be obtained with chromatography media with high efficiency, that is, small beads.

21. Gel filtration Smaller solutes retain on the column longer Therefore, retention time is inversely proportional to the size of the solute Dextran (= sephadex ) Polyacrylamide Agarose (= sepahrose ) 0.15-1.0 M salt buffer to prevent interaction of the protein with support matrix. Unlike others, protein doesn’t bind stationary phase during the process. Resolution depends on smallest possible volume of sample.

22. Also, used to determine molecular wt. of a protein. Kav = Ve - Vo/ Vt - Vo Vo = Void Volume (solvent between beads) Vt = Total Volume (pi r 2 into length) Ve = Elution Volume Shape ~ long rod shaped proteins elute at apparent molecular weights greater than their actual molecular weights. DESALTING – removing of salts from protein solutions or to carry out buffer exchanges. For e.g. Desalting columns much faster than dialysis. Spins columns.

23. Purification of tagged r-protein by column chromatography: Undergoes single step protein purification (capture step), when suitable ligand is available for protein of interest followed by second chromatographic step (polishing step) to remove remaining impurities. Affinity chromatography - on the basis of reversible interaction between protein and a specific ligand attached to a chromatographic matrix Unbound material washed away and bound target protein is by desorption. Affinity tags - highly efficient tools for protein purification. Inteins tags - to purify any r-proteins of interest in column chromatography. It excise itself and rejoin exteins with a peptide bond. Purification of protein in column chromatography is without the use of protease to remove the tag (self-cleavable proteases).

24. ION EXCHANGE CHROMATOGRAPHY Chromatography is the separation of a mixture of compounds into its individual components based on their relative interactions with an inert matrix. Ion exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to ion exchangers. This technique is extremely useful in the separation of charge compounds like proteins differing by only one charged amino acid. In ion exchange chromatography technique one can choose whether to bind the substance of interest and allow the contamination to pass through the column and vice versa. PRINCIPLE Ion exchange chromatography relies on the attraction between oppositely charged stationary phase,known as an ion exchanger and analyte. The ion exchanger consists of a inert support medium coupled covalently to positive(anion exchanger)or negative(cation exchanger) functional groups. To these covalently bound fuctional groups the oppositely charged ions are bounded (mobile counter ion),which will be exchanged with like charge ions in the sample having charge magnitude more than the ions bounded to the matrix. Thus if anion exchange chromatography is performed,negatively charged sample components will interact more with the stationary phase and will be exchanged for like charged ions already bounded to the matrix.

25. AFFINITY CHROMATOGRAPHY Affinity chromatography is a type of liquid chromatography for the separation,purification or specific analysis of sample components. It utilizes the reversible biological interaction or molecular recognition called affinity which refers to the attracting forced exerted in different degrees between atoms which cause them to remain in combination. Example: Enzyme with and inhibitor, antigen with an antibody etc. PRINCIPLE The stationary phase consists of a support system, on which the substrate(ligand) is bound covalently, in such a way that the reactive groups that are essential for binding of the target molecule are exposed. As the crude mixture of the substances is passed through the chromatography column, substances with binding site for the immobilized substrate bind to the stationary phase,while other substances is eluted in the void volume of the column. Once the other substances are eluted,the bound target molecules can be eluted by methods such as including a competing ligand in the mobile phase or changing the pH, ionic strength or polarity conditions.

26. The purity of products obtained in that way may be adequate for basic research, but additional purification steps are often required: (a)Multimeric forms and/or fragments of the recombinant protein produced by incomplete translation are also adsorbed specifically. (b)Small amounts of contaminants are adsorbed non-specifically; and (c)Losses of the ligand from the column can occur if harsh conditions of pH are required for elution.

27. MECHANISM OF AFFINITY BINDING

28. Hydrophobic interaction chromatography Hydrophobic interaction chromatography is the separation technique that separates molecules on the basis of their degree of hydrophobicity. PRINCIPLE The principle of hydrophobic interaction chromatography is based on the interaction between two molecules with hydrophobic groups. Here, the stationary phase is solid support applied with both hydrophobic and hydrophilic groups. The solvent molecules containing hydrophobic regions interact with the hydrophobic groups, thus separating them from the molecules with hydrophilic groups. The interaction is then reversed by applying an elution solution with decreasing salt gradient, which causes the molecules with hydrophobic groups to be separated from the stationary phase.

29. ( A)Highly ordered water shells surround the hydrophobic surfaces of ligand and proteins. Hydrophobic substances are forced to merge to minimize the total area of such shells[maximize entropy].Salts enhance the hydrophobic interaction. (B)The equilibrium of the hydrophobic interaction is controlled predominantly by the salt concentration.

30. REVERSE PHASE CHROMATOGRAPHY Reverse-phase chromatography is a liquid chromatography technique where the separation of molecules is achieved through hydrophobic interaction between the liquid mobile phase and the stationary phase. PRINCIPLE The principle of reverse phase chromatography is based on the interaction between two molecules with hydrophobic groups. Here, the stationary phase is solid support applied with both hydrophobic and hydrophilic groups. The solvent molecules containing hydrophobic regions interact with the hydrophobic groups, thus separating them from the molecules with hydrophilic groups. The interaction is then reversed by applying an elution solution with decreasing salt gradient, which causes the molecules with hydrophobic groups to be separated from the stationary phase.

31. DIFFERENCE

32. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY High-performance liquid chromatography is a modified form of column chromatography where the components of a mixture are separated on the basis of their affinity with the stationary phase. PRINCIPLE This technique is based on the principle of differential adsorption where different molecules in a mixture have a varying degree of interactions with the absorbent present on the stationary phase. The molecules having higher affinity remain adsorbed for a longer time decreasing their speed of movement through the column. However, the molecules with lower affinity move with a faster movement, thus allowing the molecules to be separated in different fractions. This process is slightly different from the column chromatography as in this case; the solvent is forced under high pressures of up to 400 atmospheres instead of allowing it to drip down under gravity.

33. REFERENCES https://books.google.co.in/books?hl=en&lr=&id=CN53kDLCOjAC&oi=fnd&pg=PR5&dq=recombinant+proteins+purification&ots=RQPM95z49L&sig=smJfh2qK9rkykriqQObYk0WK3x8#v=onepage&q=recombinant%20proteins%20purification&f=false https://www.researchgate.net/publication/289525226_Chromatography_-_the_essence_of_bioanalysis https://onlinelibrary.wiley.com/doi/full/10.1002/biot.201100015 https://www.sinobiological.com/resource/protein-review/protein-purification