Recombination protein production

Question 1

Define Proteins

Answer 1

The most abundant organic molecules of the living system. They have significant role in structural and functional
organization of the cell.

Question 2

Recombinant Protein

Answer 2

Once a Recombinant DNA is inserted into bacteria, these bacteria will make protein based on this rDNA.

Question 3

Two methods for recombinant proteins

Answer 3

• Molecular Cloning
• Polymerase Chain reaction

Question 4

Difference between the two methods.

Answer 4

Molecular cloning incorporates the replication of the DNA within a living cell, whereas PCR replicates DNA in the test tube, without living cells.

Question 5

Cloning process

Answer 5

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)

Question 6

What is a vector

Answer 6

Self-replicating DNA molecules used to transfer foreign DNA segments between host cells. Small in size with single restriction endonuclease site.

Question 7

What are the types of vectors

Answer 7

Plasmids, Bacteriophages and Cosmids

Question 8

Define Plasmids

Answer 8

Circular extrachromosomal molecules of DNA.
pBR322 of E.coli is most popular and widely used plasmid vector.

Question 9

Define Bacteriophages

Answer 9

Bacteriophages or simply phages are the viruses that replicate within the bacteria.
Phages can accept foreign DNA fragments of 10-20kb length.

Question 10

Define Cosmids

Answer 10

Specialized plasmids containing DNA sequence namely cos sites. Cosmids can carry larger fragments of foreign DNA compared to plasmids, 20-50 kb.

Question 11

What is a polymerize chain reaction

Answer 11

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.

Question 12

Steps in polymerase chain reaction

Answer 12

1. Denaturation
   DNA is heated to break the hydrogen bonds between the two polynucleotide strands. Two single-stranded DNA molecules serve as templates.
2. 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.
3. DNA synthesis
   The enzyme Taq polymerase is added to synthesize a complementary DNA strand.
   Lower temperature.

Question 13

Production of recombinant Insulin brief

Answer 13

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

Question 14

Applications of Recombinant DNA

Answer 14

• Hematopoietic growth factor
• Interferon
• Hormones
• Recombinant protein vaccines
• Tissue/ bone growth factors and clotting factors.
• Biological response modifiers
• Monoclonal/ Diagnostic/ Therapeutic antibodies

Question 15

Name three Hematopoietic growth factor that are of recombinant origin

Answer 15

Thrombopoietin
Erythropoietin
Ancestism

Question 16

What are antibodies

Answer 16

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.

Question 17

What are interferons

Answer 17

Infected by viruses produces protein called interferon, viral resistance to native cells.

Question 18

What are interferons

Answer 18

Infected by viruses produces protein called interferon, viral resistance to native cells.

Question 19

Types of recombinant Inteferons

Answer 19

t, alpha, beta, gamma.
Recombinant interferon alfa-2b
alfa-2a
alfa1a
1bBeta

Question 20

Technique Electrophoresis is used for?

Answer 20

Determining molecular weight of proteins

Question 21

The type of electrophoresis technique used for protein products

Answer 21

Sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Question 22

Briefly explain the electrophoresis technique

Answer 22

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.

Question 23

Importance of recombinant technology

Answer 23

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.