Selection & Screening of Recombinant cells & expression of recombinant (2) (1)
- 1. BY- SUNANDA ARYA MSc . Microbiology
- 2. • INTRODUCTION FOR SELECTION • DIRECT SELECTION OF RECOMBINANTS • INSERTIONAL INACTIVATION • BLUE- WHITE SCREENING • COLONY HYBRIDISATION • IMMUNOLOGICAL TESTS • PROTEIN EXPRESSION INTRODUCTION • MAMMALIAN PROTEIN EXPRESSION • INSECT EXPRESSION • EXPRESSION IN BACTERIA • CONCLUSION • REFERENCE
- 3. • After the introduction of recombinant DNA into the host cells, it is essential to identify those cells which received rDNA molecule - screening (or) selection. • The vector or foreign DNA present in the recombinant cells expresses certain characters or traits, while non- recombinants do not express the traits. • Transformed cells (or recombinant cells) are those host cells which have taken up the recombinant DNA molecule. In this step the transformed cells are separated from the non-transformed cells by using various methods making use of marker genes.
- 5. Methods 1- Direct selection of Recombinants 2- Insertional inactivation 3- Blue - White screening 4- Colony Hybridization 5 -Immunological Tests
- 6. • Vector designed to contain an antibiotic resistance genes. • After transformation , cells are plated on medium containing antibiotic . • Ultimately the transformed cell will grow i.e recombinants showing antibiotic resistance. Example- to clone the gene for kanamycin resistance from plasmid R6-5. • This plasmid carries genes for resistances to four antibiotics: Kanamycin, chloramphenicol, streptomycin and sulphonamide.
- 7. • In this gene the EcoRI fragments of R6-5 would be inserted into the EcoRI site of a vector such as PBR322. • The ligated mix will comprise many copies of 13 different recombinant DNA molecules, one set of which carries the gene for kanamycin resistance. • Insertional inactivation cannot be used to select recombinants when the EcoRI site of PBR322 is used. This is because this site does not lie in either the ampicillin or the tetracycline resistance of this plasmid. But this is immaterial for cloning the kanamycin resistance gene because in this case the coloned gene can be used as the selectable marker. • Transformed are plated onto kanamycin agar, on which the only cells able to survive and produce colonies are those recombinants that contain the cloned kanamycin resistance gene(c).
- 10. • Insertional inactivation is a technique used in recombinant DNA technology. In this procedure, a bacteria carrying recombinant plasmids or a fragment of foreign DNA is made to insert into a restriction site inside a gene to resist antibiotics, hence causing the gene to turn non-functional or in an inactivated state.
- 11. Three essential features for a vector est 1)An origin of replication 2)Many Unique restriction sites within selectable markers 3) At least 2 selectable markers • Upon insertion of desired gene on Tetracycline region, the gene is no more functional and the process is called insertional inactivation.
- 13. • Blue-white screening is a rapid and efficient technique for the identification of recombinant bacteria. It relies on the activity of β- galactosidase, an enzyme occurring in E. coli, which cleaves lactose into glucose and galactose. • The presence of lactose in the surrounding environment triggers the lacZ operon in E. coli. The operon activity results in the production of β-galactoisdase enzyme that metabolizes the lactose. • β-galactosidase is a protein encoded by the lacZ gene of the lac operon, and it exists as a homotetramer in its active state. However, a mutant β-galactosidase derived from the M15 strain of E. coli has its N-terminal residues 11—41 deleted and this mutant, the ω-peptide, is unable to form a tetramer and is inactive.
- 14. • This mutant form of protein however may return fully to its active tetrameric state in the presence of an N-terminal fragment of the protein, the α-peptide. The rescue of function of the mutant β-galactosidase by the α-peptide is called α-complementation. • The blue/white screening method works by disrupting this α-complementation process.
- 15. • Based on concept of Lac- operon
- 16. • If LACTOSE is there in environemnt, bacteria use this lactose & activate the lac z,y,a genes. • Lac z gene produced enzyme β-galactoisdase, which hydrolyses… LACTOSE GLUCOSE + GALACTOSE - β-galactoisdase – HOMOTETRAMER (active state) - β-galactoisdase (mutant form) –unable to form tetramer (inactive state) - MUTANT FORM NORMAL FORM - BY getting α-peptide. - The rescue of function of the mutant β-galactosidase by the α- peptide is called α-complementation
- 17. The blue/white screening method works by disrupting this α-complementation process. • The presence of an active β-galactosidase can be detected by X-gal, a colourless analog of lactose that may be cleaved by β- galactosidase to form 5-bromo-4-chloro-indoxyl, which then spontaneously dimerizes and oxidizes to form a bright blue insoluble pigment 5,5'-dibromo-4,4'-dichloro-indigo. • This results in a characteristic blue colour in cells containing a functional β-galactosidase. Blue colonies therefore show that they may contain a vector with an uninterrupted lacZα (therefore no insert), while white colonies, where X-gal is not hydrolyzed, indicate the presence of an insert in lacZα which disrupts the formation of an active β-galactosidase.
- 20. • The colony hybridization technique is based on the availability of radioactively labeled DNA probe. • A probe is radioactively labeled p32 nucleic acid (20-40 nucleotide long) with a sequence complementary to at least one part of the desired DNA. • The probe may be partially pure mRNA, a chemically synthesized oligonucleotide or a related gene, which identifies the corresponding recombinant DNA. • DNA probes have commercial significance. They diagnose specific DNA sequences (genes) and are useful in the diagnosis of diseases, microbiological tests and in research as well.
- 21. • In this technique master plates are prepared as described earlier. Replica plating of colonies is done onto a nitrocellulose filter disc which is then placed on the surface of a gelled nutrient medium and both master plate and disc are incubated to develop colonies. • Cells growing on nitrocellulose filter disc are nourished through the diffusion of nutrients from gelled nutrient medium. • The filter disc is removed and put on blotting paper soaked with 0.5 N NaOH solution. The alkali diffuses into nitrocellulose, lyses bacterial cells and denatures their DNA. Thereafter, the filter disc is neutralized by tris (hydroxymethyl) aminomethane-HCl buffer by keeping high salt concentrations. This results in binding the DNA with nitrocellulose disc in the same pattern as the bacterial colonies; to fix the cDNA properly the filter disc is baked at 80°C .
- 22. • Then it is incubated with a solution containing radioactive chemical labeled probe (p32 DNA) at suitable conditions. The probe will hybridize any bound DNA which contains sequences complementary to the probe . • By thorough washing unhybridized (unbound) probes are removed from the hybridized probe (colonies containing sequences complementary to probe) and is identified by autoradiography of the nitrocellulose filter disc . • Colonies which develop positive X-ray image are compared with master plate and picked up, and multiplied on the nutrient medium.
- 24. • The immunological techniques are the final test analogous to colony hybridization technique as described earlier. It is an alternative screening procedure which relies on expression, and generally applicable approach to identify a clone synthesizing a particular polypeptide. • This is potentially a very powerful method since the only absolute requirement is that the required mRNA encodes a protein for which a suitable antibody is available. • In the immunological test, instead of radio-labeling of DNA molecules, antibodies (immunoglobulins) are used to identify the colonies or plaques developed on master plates that synthesize antigens encoded by the foreign DNA present in plasmids of the bacterial clones. • For this purpose a special vector, known as expression vector, is designed where the foreign DNA is transcribed and translated within the bacterial cell
- 25. The growth medium containing specific anti serum may help in detection of viable immunoprecipitate (precipitin) around the colonies or plaques. The method follows :
- 26. • Broome and Gilbert (1978) purified immunoglobulin G (IgG) and bound it to an agar plate so that the antigens released through in situ lysis of bacterial colonies can bound to the fixed antibody . • The antigen is then detected by using the same IgG preparation which is radioactively labeled. This recognizes determinants on the bound antigen.
- 29. • Protein expression refers to the way in which proteins are synthesized, modified and regulated in living organisms. In protein research, the term can apply to either the object of study or the laboratory techniques required to manufacture proteins. This article focuses on the latter meaning of protein expression. However, in practical terms, recombinant protein production depends on using cellular machinery.
- 30. • Traditional strategies for recombinant protein expression involve transfecting cells with a DNA vector that contains the template and then culturing the cells so that they transcribe and translate the desired protein. Typically, the cells are then lysed to extract the expressed protein for subsequent purification. Both prokaryotic and eukaryotic in vivo protein expression systems are widely used. The selection of the system depends on the type of protein, the requirements for functional activity and the desired yield. • These expression systems are summarized in the table, include mammalian, insect, yeast, bacterial, algal and cell-free. Each system has advantages and challenges, and choosing the right system for the specific application is important for successful recombinant protein expression.
- 33. • Mammalian expression systems can be used to produce mammalian proteins that have the most native structure and activity due to its physiologically relevant environment. This results in high levels of post- translational processing and functional activity. Mammalian expression systems are the preferred system for the expression of mammalian proteins and can be used for the production of antibodies, complex proteins and proteins for use in functional cell-based assays. However, these benefits are coupled with more demanding culture conditions. • Mammalian expression systems can be used to produce proteins transiently or through stable cell lines, where the expression construct is integrated into the host genome. While stable cell lines can be used over several experiments, transient production can generate large amounts of protein in one to two weeks. These transient, high-yield mammalian expression systems utilize suspension cultures and can produce gram-per-liter yields. Furthermore, these proteins have more native folding and post-translational modifications, such as glycosylation, as compared to other expression systems.
- 34. • Insect cells can be used for high level protein expression with modifications similar to mammalian systems. There are several systems that can be used to produce recombinant baculovirus, which can then be utilized to express the protein of interest in insect cells. These systems can be easily scaled up and adapted to high- density suspension culture for large-scale expression of protein that is more functionally similar to native mammalian protein. Though yields can be up to 500 mg/L, recombinant baculovirus production can be time consuming and culture conditions more challenging than prokaryotic systems.
- 35. Baculovirus Expression System – The Invitrogen BaculoDirect Baculovirus Expression System utilizes Invitrogen Gateway technology for cloning. After a 1-hour recombinase reaction and transfection in insect cells, baculovirus containing the gene of interest is produced. A quick expression test can then be performed before amplifying the viral stock and scaling up expression. Use of this system allows for baculovirus expression in insect cells.
- 36. • Bacterial protein expression systems are popular because bacteria are easy to culture, grow fast and produce high yields of recombinant protein. However, multi-domain eukaryotic proteins expressed in bacteria often are non-functional because the cells are not equipped to accomplish the required post-translational modifications or molecular folding. Also, many proteins become insoluble as inclusion bodies that are very difficult to recover without harsh denaturants and subsequent protein-refolding procedures.
- 37. • Although eukaryotic cells can be used to express eukaryotic proteins, bacteria are simpler to grow and manipulate genetically. Therefore, it is often desirable to express eukaryotic proteins in bacteria. • Eukaryotic genes must be adapted for expression in bacteria. First, the mRNA from the gene of interest is converted to cDNA to provide uninterrupted coding DNA. The cDNA is cloned between a bacterial promoter and a bacterial terminator so the bacterial transcription and translation machinery express the coding sequence.
- 39. • In production of recombinant protein, the gene for the protein of interest is cloned into a vector and expressed into protein in a model organism. • The plasmids of the copy of the interest gene, or the expression vectors, are often used to enhance gene expression. These vectors provide a strong promoter to drive expression of the cloned gene. Expression vectors also contain genes for antibiotic resistance to allow selection of the vector and the recombinant protein. • Expression vectors are used to make eukaryotic proteins in bacteria. The vector has the ribosome-binding site, terminator sequences, and a strong regulated promoter. The eukaryotic gene is a cDNA copy of the mRNA. Once cloning is completed, plasmids are taken up into competent cells (chemically competent or electro competent E. coli) for propagation and storage, by a process called transformation.
- 40. • Because bacteria cannot process introns so it is standard procedure to clone the cDNA version of eukaryotic genes, which lacks the introns and consists of uninterrupted coding sequence. And the cDNA version of eukaryotic genes is generally used, even for expression in eukaryotic cells.
- 41. • Prescott's Microbiology, 10th Edition by Joanne Willey and Linda Sherwood and Christopher J. Woolverton. • Overview of Protein Expression. Thermofisher.scientific.com • Expression of recombinant proteins in insect and mammalian cells. Article .sciencedirect.com • www.ncbi.com • Slideshare. Selection and screening of recombinant clones.neeru02.