G: Questions

Question 1

In a recombinant expression host, how may induction of a heterologous gene be controlled by temperature?

Answer 1

The leftward (PL) promoter from bacteriophage lambda is controlled by the cI repressor. A temperature-sensitive mutant of the ci repressor (CI857) is used to carry out PL-directed transcription. If cells are cultivated at 30C, then the cI repressor will be active. When the cell culture reaches the required cell density (biomass), the temperature is increased to 42C, inactivating the repressor, and allowing transcription to start. This gets over the need for the use of an expensive chemical inducer.

Question 2

Provide two advantages relating to the use of E. coli as an expression host for a recombinant DNA protein?

Answer 2

Genetics and molecular biology well understood; cultivation know-how is well-developed; fast doubling time (20-30 minutes)

Question 3

List any two disadvantages of E. coli as an expression host for recombinant NDA biopharmaceuticals?

Answer 3

Intracellular localisation of heterologous proteins; inclusion bodies; no glycosylation; LPS endotoxin; grows naturally in human gut.

Question 4

What is human insulin emp?

Answer 4

Humanised porcine insulin manufactured by proteolytic removal of an 8 amino acid segment from the B-chain and replacement with a synthetic octapeptide that bears the correct amino acid residue at position 30.

Question 5

Briefly, describe how porcine insulin may be made to look like the human form of this?

Answer 5

‘Human insulin emp’: the single different amino acid in the porcine version (B 30 alanine residue) was cut out of the molecule and replaced with a human version (threonine). Subjecting the porcine insulin to proteolytic digestion with trypsin result in a single cleavage of the peptide bond linking arginine 22 and glycine 23 in the insulin B chain. This produces a shortened insulin molecule and a B chain terminal octapeptide. These products can be separated on a gel filtration column. A synthetic octapeptide bearing the correct amino acid residue at position 30 is then grafted onto the shortened insulin to yield a semi-synthetic human insulin product.

Question 6

What is an inclusion body, and how may their formation be reduced?

Answer 6

Overexpression of heterologous proteins in E. coli results in the formation of insoluble tangles of protein. An inclusion body is an intracellular body comprising a tangle of recombinant protein, necessitating recovery by denaturation and refolding into an active form of the protein.
Growth of the cell at 30C rather than 37C; use of lower copy number plasmid for expression; expression as fusion protein with hydrophilic component.

Question 7

What is a heterologous protein?

Answer 7

A (recombinant) protein not produced naturally within a cell.

Question 8

Provide two disadvantages relating to the use of E. coli as an expression host for a recombinant DNA protein?

Answer 8

a. Largely intracellular localization of homologous protein (necessitating cell disruption and subsequent downstream processing);
b. It often forms insoluble inclusion bodies with high level expression, necessitating unfolding and refolding of the protein into the correct shape to yield activity;
e. Grows naturally in the human gut (and so in retrospect, an unwise choice for a large scale production organism). This necessitates using a ‘weakened’ strain (usually induce a mutation to auxotrophy).
f. No proteolytic cleavage of proproteins.

Question 9

What are inclusion bodies?

Answer 9

Many recombinant proteins when tangled aggregates produced when recombinant proteins are unable to undergo proper folding within the cell due to high expression levels in their expression host

Question 10

Dissolving inclusion bodies

Answer 10

Inclusion bodies can be dissolved in solutions of strong chaotropes in the presence of a reducing agent

Question 11

After dissolving inclusion bodies

Answer 11

The dissolved protein is then allowed to refold to a native conformation by gradually removing the chaotropic agents via diafiltration/chromatography, and diluting the solution down to a total protein concentration of 20-100 mg/L

Question 12

Only advantage of inclusion bodies

Answer 12

difference in density between IB and cell fragments means that they can be easily separated by centrifugation after cell breakage. The resultant IB paste can be stored frozen for several months, providing manufacturing flexibility. The IB is virtually pure recombinant protein (about 90%), and can also protect the protein against unwanted proteolytic degradation

Question 13

Disadvantages of human therapeutic protein production by rDNA technology in animal cells

Answer 13

a. Complex nutritional requirements (costly);
b. Potential contaminants from growth medium ingredients (such as prions from foetal calf serum);
c. Slow growth;

Question 14

Upstream processing

Answer 14

refers to the initial fermentation process that results in the generation of raw product (typically culture fluid)

Question 15

Downstream processing

Answer 15

refers to purification of the final product and generation of finished product format

Question 16

Insulin - what and functions

Answer 16

polypeptide hormone produced in the β-cells of the islets of Langerhans in the pancreas

1. It allows glucose to pass into cells, where it is used for energy;
2. It suppresses excess production of sugar in the liver and muscles;
3. It suppresses the breakdown of fat for energy.

Question 17

acidosis

Answer 17

Without glucose for energy, the body begins to metabolize protein and fat. Fat metabolism results in the production of ketones in the liver. Ketones are excreted in the urine along with sodium bicarbonate, which results in a decrease in the pH of the blood

Question 18

type 1 or juvenile-onset diabetes

Answer 18

Failure of the body to make sufficient insulin results in insulin-dependent diabetes mellitus (type 1 or juvenile-onset diabetes). This may be controlled by parenteral (subcutaneous injection) administration of insulin.
The disease is characterized by hyperglycaemia

Question 19

type II or ‘maturity onset’ diabetes

Answer 19

Non-insulin-dependent (type II or ‘maturity onset’) diabetes results from a combination of insulin resistance or β-cell insulin secretory dysfunction; highest. It can often initially be managed by exercise and modified diet.

Question 20

gestational diabetes

Answer 20

a state of carbohydrate intolerance which can develop in pregnancy.

Question 21

preproinsulin

Answer 21

Although a dimer as a mature molecule, insulin is initially synthesized in the body as a single polypeptide precursor (108 amino acids and a 23-amino acid signal peptide (hydrophobic) at its N-terminus)

Question 22

proinsulin

Answer 22

prodcued by removal of signal peptide in protein trafficking

Question 23

mature insulin production

Answer 23

made from proinsulin by proteolytic cleavage in secretory granules (this produces insulin and the 34-amino acid connecting C peptide - which is further proteolytically modified by the removal of a dipeptide from each end)

Question 24

mature insulin

Answer 24

comprises 2 polypeptide chains connected by 2 inter-chain disulfide linkages (molecular mass 5,807 Da). The A chain contains 21 amino acids and the B chain 30 amino acids

Question 25

Modern insulins

Answer 25

derived from animals are subjected to further chromatography steps to remove impurities. Such insulins are purified on Sephadex G-50 columns to produce a ‘single-peak’ insulin (but these are not homogeneous preparations).

Question 26

pI of insulin

Answer 26

pH 5.4

Question 27

insulin administration

Answer 27

parenteral administration (by sub-cutaneous injection (daily, also ADRs common - looks ‘foreign’)

Question 28

human insulin crb

Answer 28

Approach to express chemically synthesized genes for the A and B chains of insulin in 2 different E.coli cells as chimaeric fusion proteins (to aid stability) of either β- galactosidase (from lac operon) or tryptophan synthetase (from trp operon) (and incorporating a methionine residue susceptible to cleavage by CnBr), and to purify the chains separately.

The separate chains were then clipped away from the β-galactosidase precursor by treatment with CnBr, purified, and then incubated together under oxidizing conditions to promote inter-chain disulfide bond formation. This produces ‘human insulin crb.

Question 29

human insulin prb

Answer 29

Approach to express the nucleotide sequence for human proinsulin in E. coli as a Trp-E fusion protein. The plasmid used to express proinsulin contained the trp promoter-operator, featured a deletion in the attenuator region of the leader peptide, and encoded the sequence for the amino terminal segment of the trp E gene product, which leads to the production of proinsulin as a fusion gene product termed ‘LE’ (and containing a methionine residue between the proinsulin and the anthranilate synthetase sequence).

This is then chemically cleaved from the E. coli carrier protein by treatment with CnBr and purified. The primary structure of proinsulin contains all the information needed for correct folding and the C peptide can be excised in vitro to produce mature insulin. These preparations are termed ‘human insulin prb’.

Question 30

New Insulin

Answer 30

1. A conventional insulin (such as Humulin R produced in E. coli or Novalin R produced in Saccharomyces cerevisiae) shows a peak after 2-3 hours with a duration of action lasting over 6 hours.
2. Apidra (insulin glulisine, Sanofi Aventis) has a B3 asparagine residue replaced by lysine and a B29 lysine replaced by glutamic acid. This is a rapid-acting insulin.
3. Insulin Aspart (NovoLog, Novo Nordisk) is another fast-acting insulin made via site directed mutagenesis: proline (B28) has been replaced by an aspartic acid residue, causing increased electrostatic charge repulsion and preventing the formation of multimers.

Also: Insulin Lispro, Levemir and Lantus