Synthetic Insulins

Question 1

describe the chemical structure of monomeric human insulin

Answer 1

1. a heterodimer- has 2 chains linked by disulphide bonds
2. gene encodes a 138 amino acid precursor protein (preproinsulin)
3. Human insulin is comprised of two polypeptide chains, The A chain has 21 amino acids and the B-chain 30.
4. The molecular weight of monomeric insulin is about 5kDa and the mature protein is not glycosylated

Question 2

describe the processing of the insulin protein

Answer 2

1. signal sequence of precursor polypeptide cleaved during transfer to ER
2. this gives second precursor (85 aa proinsulin)
3. proinsulin converted to insulin by further proteolysis
4. mature insulin molecule consists of 2 polypeptide chains joined by disulphide bonds (51aa)

Question 3

what are the characteristics of mature human insulin

Answer 3

1. small molecule
2. no evidence of N linked glycosylation
3. heterodimer- A and B chain
4. internal S-S bond in A chain
5. A and B chain linked by 2 disulphide bonds
6. mature insulin forms hexamers

Question 4

give examples of second generation recombinant insulins

Answer 4

1. rapid (analogue)- aspart, lispro
2. short (soluble)
3. long- detemir, glargine
4. mixtures (biphasic)- biphasic aspart

Question 5

what is mutagenesis of cloned DNAs and how can this be achieved

Answer 5

1. to convert native insulin into analogues, genetic engineering is used to introduce the desired alterations into the cloned gene
2. single or small numbers of nucleotides in the insulin gene can be changed by site directed mutagenesis
   - can also be done by PCR
3. if several changes plus codon optimisation is required, can get the new gene synthesised commercially

Question 6

describe how site directed mutagenesis works

Answer 6

1. involves primer with mutation and plasmid with cloned gene
2. DNA polymerase to copy full plasmid and return to double stranded state
3. DNA replication will create a mixture of mutant and wild type plasmids

Question 7

describe how gene modification by PCR works

Answer 7

1. include mutations on one side of the primers in order to produce a modified pCR product
2. extremely easy when the changes are to be introduced at the end of the gene

Question 8

describe how codon optimisation is used

Answer 8

1. human genes encoding therapeutic proteins are rarely expressed in human cells
2. for insulin, bacterial or yeast cells are used
3. but heterologous gene expression in these hosts gives reduced yields because tRNA pools vary between organisms
4. DNA code is degenerate
   - by adjusting codons to match the host tRNA abundance, yields are improved
5. so for the human insulin gene, codon usage is altered to favour the codon preferences for protein synthesis in E coli, or Pichia pastoris

Question 9

what was the 1st recombinant therapeutic protein

Answer 9

humulin

Question 10

what does crb stand for

Answer 10

chain recombinant bacterial

Question 11

what does prb stand for

Answer 11

proinsulin recombinant bacterial

Question 12

give examples of the problems of the crb recombinant insulin

Answer 12

low yields, cross linking of strands inefficient, downstream processing incurred large losses of product

Question 13

how were the problems of crb recombinant insulin resolved

Answer 13

development of prb
- produce proinsulin and cleave central peptide enzymatically

Question 14

what are the advantages of recombinant insulin

Answer 14

1. cheap to manufacture, 98% purity
2. by applying genetic engineering technology, modified forms can be produced with different release profiles

Question 15

describe the production of insulin in yeast

Answer 15

1. required modification of proinsulin gene construct
2. allows efficient expression in Saccharomyces cerevisiae
3. proinsulin construct modifications- fusion of native A chain to B chain lacking the C terminal B30 threonine
4. construct fused with a-factor signal sequence of S. cerevisiae for secreted expression- easier to purify
5. high yield

Question 16

describe the production of insulin in Pichia pastoris

Answer 16

1. attains high cell densities in fermenter
2. insulin production controlled b alcohol oxidase 1 promotor
3. add methanol to switch production on
4. very high yields

Question 17

what are the advantages of producing insulin from transgenic plants

Answer 17

• cost effective
• high quality protein processing
• absence of human pathogens
• ease of production and presence of eukaryotic machinery for post translational modifications

Question 18

what is the active form of human insulin

Answer 18

The 5kDa monomeric form

Question 19

What modifications are made to “normal” human insulin to create the fast-acting analogue, Lispro

Answer 19

Two amino acids (lysine29 and proline28) residues at the C-terminal end of the B chain are REVERSED (Proline now aa 29 and lysine now aa 28).
This does not alter receptor binding, but minimizes the tendency to of normal monomeric insulin to form dimers (due to reduced hydrogen binding) and then hexamers

Question 20

Why does heterologous gene expression often provide reduced yields of product?

Answer 20

Because the tRNA pools differ between organisms

Question 21

how can the yields be increased

Answer 21

if the human insulin gene is CODON-OPTIMISED for expression in E.coli
This is most easily achieved using a syngene

Question 22

Which plants have been engineered to produce recombinant human insulin

Answer 22

tobacco, lettuce and mouse ear cress