recombinant DNA

Question 1

Drugs based on recombinant DNA technology are expanding in availability and are being used as therapeutic agents. e.g.?

Answer 1

insulin, human growth hormone, hepatitis B vaccine and interferon.

Question 2

Recombinant DNA alters the DNA of the host and has found application in a number of disciplines. for instance?

Answer 2

For instance, for the medical discipline, it is possible to use this technology to produce insulin. Another example is of small molecule drugs, a suitable precursor for production of artemisinin is now possible to satisfy world demand for this antimalarial and its derivatives. (Genetically modifying yeast has been shown to make the bio-synthetic intermediate (S)-reticuline – morphine precursor.)

Question 3

Simplified appreciation of recombinant DNA and application of the technology, explain the diagram in that slide

Answer 3

see in slide 3

Question 4

Recombinant DNA, also called rDNA or chimera, involves a number of steps.
In summary?

Answer 4

Use restriction endonucleases (also referred to as restriction enzymes) to cleave off the required
1. segment. May require to produce more of the segment by means of polymerase chain reaction (PCR).

2. Select a suitable cloning vector (e.g. plasmid), DNA fragment that is able to replicate e.g. from
   bacteria, yeast and viruses.
3. Using DNA ligase covalently link the segment to the cloning vector by reforming the sugar
   phosphate backbone and now we have ‘recombinant DNA’.
4. Insert the rDNA into a host which will already have the system for DNA replication.
5. Select host cells that have the rDNA.

‘Recombinant DNA technology, joining together of DNA molecules from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry.’

Question 5

explain the diagram in slide 5

Answer 5

see in slide 5

Question 6

Restriction enzymes recognise ________________________

Answer 6

Restriction enzymes recognise specific base sequences where they will make the cut (recognition site). They occur in many bacterial species and have a protecting role e.g. viral infection. The host, its DNA is protected from the enzymes by methylation of the DNA by DNA methylase. Three types of restriction enzymes are recognised.

Question 7

Type II are _____________

Answer 7

more selective and will cleave at particular phosphodiester bonds at the recognition site.

Question 8

A particular segment of DNA of interest, after cleavage with restriction enzymes, can be _________________

Answer 8

isolated and purified by means of electrophoresis in agarose gel. The cloning vector DNA (e.g. plasmid) would similarly be digested by using the same restriction enzyme (base pair sequence has to match).

Question 9

The link between the cloning vector and the DNA segment is made by means of _______________

Answer 9

DNA ligase, by formation of a new phosphodiesterase bonds using ATP. Complementary sticky ends facilitate the ligation reaction.

Question 10

Type I cleave DNA at _________

Answer 10

random sites.

Question 11

Type III cleave DNA about ______________

Answer 11

25 base pairs from the recognition site.

Question 12

The cloning vector DNA (e.g. plasmid) would similarly be _______________

Answer 12

digested by using the same restriction enzyme (base pair sequence has to match).

Question 13

particular segment of DNA of interest, after cleavage with restriction enzymes, can be isolated and purified by means of electrophoresis in _______________

Answer 13

agarose gel.

Question 14

Expression vectors are used to _________________

Answer 14

improve the rate of expression of the cloned gene. This involves replacing certain regulatory elements (e.g. promotors [which can be nutritional] and transcription termination sequences) in the vector. This can be used to produce more of the desired proteins e.g. insulin.

Question 15

Plasmids are circular DNA and replicate separately to the host’s chromosomes. They use _____________

Answer 15

the cells resources for their purpose of replication and gene expression. They can be regarded as a kind of parasite, but these tend to be more of a symbiotic existence. Some plasmids have genes that express for certain antibiotic resistance. This can be used to identify plasmids that have the recombinant version (see diagram above). The plasmids with the recombinant DNA can be introduced into bacterial cells (e.g. E coli) by incubation at varying temperatures in CaCl2 solution.
Other cloning vectors include e.g. yeast, microalgae, insects and mammalian cells, but bacteria are used often for protein expression. See morphine lecture and malaria case study notes for production of suitable intermediates using yeast.

Question 16

Expression vectors are used to ________________

Answer 16

improve the rate of expression of the cloned gene. This involves replacing certain regulatory elements (e.g. promotors [which can be nutritional] and transcription termination sequences) in the vector. This can be used to produce more of the desired proteins e.g. insulin.

Question 17

Applications of rDNA are wide and spreading further with possibility of treating DNA aberrations that cause disease (e.g. cystic fibrosis). Current applications include?

Answer 17

Food Industry: Chymosin enzyme (normally in rennet) is produced in large quantities and more cheaply.

Agricultural Industry: rDNA with some crops so that they are tolerant to herbicides which then affects only weeds.

Medical Research: Development of diagnostic technique for HIV based on antibodies.

Algae proteins partially restore man’s sight

Question 18

The technique is based on proteins, produced in algae, called _____________which change their behaviour in response to light. The microbes use them to move towards the light.

The first step in the treatment was gene therapy. The genetic instructions for making the ____________ were taken from _________ and given to cells in the deep surviving layers of the retina at the back of his eye.
Algae proteins partially restore man’s sight

Answer 18

channelrhodopsins,

rhodopsins

algae

Question 19

what are the applications in pharmaceutical industry

Answer 19

Produce blood clotting factor VIII in large quantities rather than rely on donated blood for
treatment of Haemophilia A patients.

rDNA to prepare human insulin rather than rely on animal sources e.g. Aspart®. The process
involves the isolated human insulin gene linked to a DNA vector and then replicated in host
organisms, either Escherichia coli or Saccharomyces cerevisiae. The gene is expressed in the host
cells and human insulin is produced in large amounts in a short period of time. Besides insulin and
factor VIII, there are other recombinant proteins produced by this method e.g. erythropoietin,
botulinum toxin (Botox), papain, collagenase and streptokinase.

Human growth hormone for treatment of patients with defective pituitary gland rather than rely
on human cadavers.

rDNA of the hepatitis B virus surface antigen (produced with the use of yeast cells) for production
of hepatitis B vaccine, used for preventing infection of the liver by the virus.

Question 20

what are the Applications of Recombinant Proteins for Therapeutic Use

Answer 20

Leader et al (2008) proposed that protein therapeutics are grouped according to function.

Group 1: Protein therapeutics with enzymatic or regulatory activity.

1a Replacing a protein that is deficient/abnormal e.g. Insulin, Factor VIII
1b Augmenting an existing pathway e.g. Erythropoietin, G-CSF, GM-CSF, IL-11
1c Providing a novel function/activity e.g. Botulinum toxin type A (Botox), Papain, Collagenase,
Streptokinase

Group 2: Protein therapeutics with Special Targeting Activity (Monoclonal antibody, mAb/mab)

2a Interfering with a molecule or organism e.g. Trastuzumab (Herceptin), Rituximab (Rituxin), Infliximab
(Remicade), Muromonab-CD3 (OKT3)
2b Delivering other compounds or proteins e.g. Denileukin diftitox (Ontak)
2c Treatment for Alzheimer’s disease, Aducanumab (2021)

Group 3: Protein Vaccines

3a – Protecting against a deleterious foreign agent e.g. HBsAg (Recombivax HB), HPV Vaccine
(Gardasil)
3b – Treating an autoimmune disease e.g. Anti-Rhesus (Rh) immunoglobulin G (Rhophylac), Rh
prophylaxis
3c – Cancer Vaccines – in trials

Group 4: Protein Diagnostics (Monoclonal antibody, mAb/mab)

Used in the diagnosis of a range of disorders,
E.g. TSH (Thyrogen), Arcitumomab (CEA-scan), Nofetumomab (Verluma)

Arcitumomab - Widely used for cancer imaging and therapy.

Question 21

explain ADEPT

Answer 21

‘Antibody Directed Enzyme Prodrug Therapy’.

MAb-enzyme conjugate is administered.
Prodrug is given.
At target site, prodrug is activated by enzyme activity.
Enables toxic drug to accumulate within tumour at high concentration.
Enzymes have high substrate turnover rate generating many drug molecules.
Recently, an anti-erbB2 dsFv-b-lactamase fusion protein was designed to activate cephalosporin-based prodrugs (cephalosporin-doxorubicin).

Question 22

explain insulin as an example of ADEPT

Answer 22

EXAMPLE – INSULIN:

First recombinant protein licensed for therapeutic use.
Insulin instructs tissues to respond to glucose concentrations via insulin receptor
Insulin is a monomer of two polypeptide chains:

A chain-21 amino acids
B chain-30 amino acids

Synthesised in islets of Langerhans as preproinsulin.
Diabetes affects 40-80 million.

Treatment uses recombinant human or porcine or bovine sources

There are 2 problems with therapeutic insulin:

Delivery into bloodstream is fatal ! (hypoglycaemia).
Time lag between injection and uptake in blood stream.

Question 23

what is the reason for time delay in slide 14

Answer 23

Reasons for Time Delay:
Sluggish uptake due to oligomersation, insulin forms dimer, hexamers etc due to synthesis and on storage, monomers are the active form, 0.1% of regular insulin is in monomeric form and therefore active.
Use genetic engineering to generate short-acting analogues.
Aggregation is due to storage process.

Engineer a form of insulin that is:

a) Active
b) Stable
c) Monomeric

Question 24

state facts about engineered insulin

Answer 24

Based on a knowledge of the monomer:monomer interface.
This required good structural information.
The crystal structure of the insulin hexamer was available at 1.5 Å resolution.
From this information about the nature of the monomer:monomer interface was obtained.
Surfaces at the interface were almost perfectly complementary.
Interactions stabilising the dimer were hydrogen bonds, electrostatic interactions and van der waals interactions.

Question 25

what are the Two strategies which were applied to overcome the problem of aggregation between the monomers:

Answer 25

i) Increase steric bulk in the dimer interface

ii) Induce repulsion between surfaces.