Recombination protein production Flashcards
Define Proteins
The most abundant organic molecules of the living system. They have significant role in structural and functional
organization of the cell.
Recombinant Protein
Once a Recombinant DNA is inserted into bacteria, these bacteria will make protein based on this rDNA.
Two methods for recombinant proteins
- Molecular Cloning
- Polymerase Chain reaction
Difference between the two methods.
Molecular cloning incorporates the replication of the DNA within a living cell, whereas PCR replicates DNA in the test tube, without living cells.
Cloning process
Gene of interest is cut out with restriction enzymes (RE)
Host plasmid (circular chromosome) is cut with same RE
Gene is inserted into plasmid and ligated with ligase.
New (engineered) plasmid inserted into bacterium (transform)
What is a vector
Self-replicating DNA molecules used to transfer foreign DNA segments between host cells. Small in size with single restriction endonuclease site.
What are the types of vectors
Plasmids, Bacteriophages and Cosmids
Define Plasmids
Circular extrachromosomal molecules of DNA.
pBR322 of E.coli is most popular and widely used plasmid vector.
Define Bacteriophages
Bacteriophages or simply phages are the viruses that replicate within the bacteria.
Phages can accept foreign DNA fragments of 10-20kb length.
Define Cosmids
Specialized plasmids containing DNA sequence namely cos sites. Cosmids can carry larger fragments of foreign DNA compared to plasmids, 20-50 kb.
What is a polymerize chain reaction
A method for amplifying DNA segments using cycles of denaturation, annealing to primers, and DNA polymerase-directed DNA synthesis.
PCR copies a DNA molecule without restriction enzymes, vectors, or host cells.
Faster and easier than conventional cloning.
Steps in polymerase chain reaction
- Denaturation
DNA is heated to break the hydrogen bonds between the two polynucleotide strands. Two single-stranded DNA molecules serve as templates. - Annealing
Short nucleotide sequences (primers-20-30 nucleotide long for DNA replication) are mixed with the DNA and bind to complementary regions on single-stranded DNA. - DNA synthesis
The enzyme Taq polymerase is added to synthesize a complementary DNA strand.
Lower temperature.
Production of recombinant Insulin brief
1) DNA sequence of insulin for two chains A and B are synthesized and separately inserted into two PBR322 plasmid vector.
These gene are inserted by the side of β-galactosidase gene of the plasmid.
2) The recombinant plasmid were then separately transformed into E. coli host.
3) The recombinant host produced pro-insulin chains. ie, fused β-galactosidase-A chain and β-galactosidase-B-chain separately.
4) These pro-insulin chains A and B were separated from β-galactosidase by treatment with cyanogen bromide.
5) After detachment, A and B chains are purified.
6) Then joined invitro to reconstitute the naïve insulin by sulphonating the peptide chains with sodium disulphonate and sodium sulphite
Applications of Recombinant DNA
- Hematopoietic growth factor
- Interferon
- Hormones
- Recombinant protein vaccines
- Tissue/ bone growth factors and clotting factors.
- Biological response modifiers
- Monoclonal/ Diagnostic/ Therapeutic antibodies
Name three Hematopoietic growth factor that are of recombinant origin
Thrombopoietin
Erythropoietin
Ancestism
What are antibodies
Antibodies are immune system related proteins called immunoglobulins. Each antibody consists of four polypeptides– two heavy chains and two light chains joined to form a “Y” shaped molecule. Antibodies are divided into five major classes, IgM, IgG, IgA, IgD, and IgE.
What are interferons
Infected by viruses produces protein called interferon, viral resistance to native cells.
What are interferons
Infected by viruses produces protein called interferon, viral resistance to native cells.
Types of recombinant Inteferons
t, alpha, beta, gamma.
Recombinant interferon alfa-2b
alfa-2a
alfa1a
1bBeta
Technique Electrophoresis is used for?
Determining molecular weight of proteins
The type of electrophoresis technique used for protein products
Sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Briefly explain the electrophoresis technique
Proteins are denatured by boiling in the SDS solution. All charges of protein are masked by negative charge of dodecyl sulphate.
Protein moves on polyacrylamide gel strictly on basis of size of protein molecule.
For visualization of proteins on the gel reagents used are silver nitrate, coomassie brilliant blue dye.
Importance of recombinant technology
Used in the production of insulin, human growth hormone, vaccines, interferons.
Used in medical applications, medications and vaccines.
Development of improved drug delivery system.
Allows introduction of modifications into proteins at desired positions.
