Production of Human Insulin

using
rDNA Technology

Expression vectors
To produce the product of a cloned gene for further studies
1)

Expression vectors with a strong promoter

More mRNA
2)

More protein

Expression vectors with an inducible promoter

Foreign proteins when overexpressed could be toxic
Keep the gene expression off till it is time to turn it on

a.
b.
3)

Drug-inducible (e.g. IPTG or arabinose)

Heat-inducible

Expression vectors with a fusion tag for affinity purification

Facilitate the purification of the expressed protein

1)
2)
3)

6 Histidine tag
Glutathione transferase tag (GST)
Maltose-binding protein tag

The pharmaceutical industry produces a wide range of

products using recombinant DNA techniques
Human proteins can be
cloned
Gene recovered from
human genome (as
genomic DNA or cDNA)
Placed in an expression
vector using ligation
techniques and
restriction
endonucleases
Transform bacteria (e.g.
Escherichia coli or
Bacillus spp.)
Grow in batch culture in
large industrial scale
vats
Purify protein product

Fusion Tags
Tag

Purification

Commercial vectors

6-10 his tag

Ni column (IMAC)
immobilized metal affinity
chromatography

pPRO, pBAD, PET

GST(Glutathione S
Transferase)

glutathione

MBP (maltose binidng

protein

Maltose column

Tags can be removed by including a protease cleavage site: Factor Xa, thrombin,
enterokinase

Therapeutics
Human proteins produce fewer side effects
than proteins from other animals (e.g. pork
insulin vs. human insulin)
Hormones or hormone-like compounds are
used for human therapy
Enzymes
Antisense RNA

A. Insulin
Made by pancreatic beta cells
Enables cells to take up glucose from the bloodstream to
use in production of ATP
Insufficient insulin causes diabetes (insulin dependent
diabetes mellitus IDDM)
Must inject insulin to avoid physiological complications
Cells cannot take up glucose
Insufficient ATP is made
Glucose spills into urine (excreted by kidneys; kidney
tries to dilute glucose by excreting large amounts of
water)

 Complications of diabetes (60 x 106 worldwide cases)

Retinopathy
Kidney failure
Nerve disorders
Circulatory diseases (including gangrene and stroke)
Diabetic coma (pH imbalance caused by fat metabolism
producing ketones)

Frederick Sanger
In 1958 british molecular biologist
Frederick Sanger determined the
primary structure of insulin. It
was the first protein to have its
sequence be determined. He
was awarded the Nobel Prize in
Chemistry for his work.

Structure of Insulin
Insulin is made up of 2 chains = 51 amino acids total
A chain = 21 amino acids
B chain = 30 amino acids

S - S
__l____________l____ A
S
S
___S___________S____________________ B

 Two disulfide bonds hold A and B together (interchain disulfide bond)

One disulfide bond within the A chian (intrachai disulfide bond)
Insulin processing
Preproinsulin
Contains a signal sequence + 3 sections of amino acids
Signal sequence is removed after targeting to RER
Translation continues on RER > forming proinsulin
Removal of 33 amino acids at Golgi Apparatus, and
joining of A and B chains to form insulin
Preproinsulin too difficult for E. coli to produce
Simplification had to be made!

Recombinant expression of Insulin

 Before recombinant insulin was available, insulin was

obtained from cows or pigs pancreases (7-10 lb pancreatic
tissue per patient per year)
Cow (Bovine) = 3 amino acid differences
Pig (Porcine) = 1 amino acid difference
Amino acid differences can stimulate allergic responses
Therefore human insulin is preferred

B. Human Growth Hormone (HGH)

HGH promotes overall body growth by increasing: amino acid uptake
by cells, protein synthesis and fat utilization for energy
Dwarfism caused by insufficient production of HGH by the pituitary
gland
Growth retardation
Chubby face
Unusual body properties as an adult
~ 4 feet tall only
IQ = Normal
HGH can treat dwarfism to help undersized children reach their
normal height and size

Recombinant Expression of Human growth hormone

Old method

Purification of HGH from cadaver pituitary glands

8 cadavers/year for 8 10 years per patient
Risky to use brain tissue
Prion disease transmission: Creutzfeldt-Jacob Disease (CJD)
Muscle wasting
Convulsions
Tremors
Dementia
24 cases reported by 1993 in France from cadaver HGH

New method
Gene production for HGH synthesis
Protropin Genentech
Humatrope Eli Lilly

Recombinant Expression of Hepatitis B