Biology And Physiology

Question 1

Give named examples of recombinant human proteins and describe their clinical and reasearch applications

Answer 1

Recombinant Insulin, Growth hormone, Clotting factors and metabollic enzymes can all be used to replace defective proteins in endocrine and metabolic disorders

Recombinant Growth hormone receptor inhibitors can be used to interfere with harmful molecular proteins produced by the body

Recombinant erythropoietin can be used to enhance/augment existing pathways

Question 2

Outline the benefits of using rDNA technology to produce purified
human proteins for research or therapeutic uses

Answer 2

rDNA is Useful as proteins are very complex molecules with highly specific functions that are difficult to replicate in chemical drugs.

They can also therfore produce human proteins from non-human sources

They can improve stability, yield or ADME properties

Reduced possibilty of immune rejecion or allergic reaction which could occur when obtained from a non-human source

Question 3

Give examples of how genetically engineered modifications to protein
sequences (e.g. insulin, EPO, cerezyme) can alter activity or ADME properties

Answer 3

Erythropoietin is a growth factor which stimulates erythrocyte production in bone marrow.

It is used to treat chronic anaemia in CKD. It is a glycosylated protein with a half life of 5 hours in the blood, by a change of 2 amino acids introduces 2 additional N-glycosulated sites which increases the half life by 3 fold meaning fewer injections would be required

Question 4

Describe problems/ barriers encountered with producing human proteins
using bacterial expression systems

Answer 4

• Human genes contain introns and exons which do not exist in bacteria so they can not process the introns and remove them
  
  • So we remove intron sequences using cDNA(copy of processed RNA)
• Bacterial RNA polymerase can not recognise mammalian promoter sequence
• To achieve this we use Reverse transcriptase to make cDNA from an RNA template
  
  • This is the first strand synthesis
    
    • We also have to degrade the mRNA so we are only left with the cDNA,
    • We can then use a DNA polymerase and a random primer to generate the second strand which can thus be use

Question 5

Be able to describe the features of other expression systems e.g yeast, plant,
or mammalian cells, or transgenic plants and animals (GMOs)

Question 6

Be able to define what a GMO is and discuss the potential risks/ hazards and
Containment requirements associated with production of GMOs

Question 7

Be able to describe different host/vector expression systems, bacterial plasmids,
inducible promoters and their uses

Question 8

Be able to name/describe genetic engineering toolkit components such as
Restriction enzymes, DNA ligase, PCR (Taq Polymerase), Reverse transcriptase,
Site-directed mutagenesis, DNA sequencing and how they are used.

Question 9

Be able to describe in detail the the steps involved in cloning and bacterial
expression of a human protein, starting with mRNA from human tissue.

Question 10

Describe methods used to purify and detect recombinant proteins including
inducible expression, epitope tags, western blots, affinity purification