Proteins

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Proteins

• Most of the biotech drugs are proteins or
  peptides
• Amino acids are the building blocks of proteins
• The genetic code specifies 20 different amino
  acids

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Protein Structures
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Protein Folding and Modifications

• Protein folding
• Proteins must be properly folded for function
• Unfolded proteins are insoluble and unstable
• Post-translation modifications
• May be required for protein function -case by
  case basis
• e.g., phosphorylation, glycosylation

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Protein Folding
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Protein Folding Gone Bad
Expression in E. coli may lead to inclusion
bodies improperly folded or unfolded
proteins Can use detergents to stabilize and
allow protein to fold basically unfold and then
allow protein to fold In humans..can lead to
disease state
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Alzheimers Plaques and Tangles
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Alzheimer Amyloid Production
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Refolding - Sticky Proteins - Aggregates
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Glycosylation
Most extensive post-translational modification
made by eukaryotic cells. Addition of
carbohydrates (mannose, glucose, galactose) to a
reactive side chain of an amino acid.
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Why do proteins need to be glycosylated?
Involved in protein interactions Helps to
solubilize and stabilize proteins Effects protein
biosynthesis and secretion Immune protection For
many glycoproteins Full glycosylation full
biological activity
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Glycosylation
Recombinant proteins expressed in eukaryotic
cells will be glycosylated. E. coli does NOT
glycosylate proteins.
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Glycosylation
N-linked glycan Amide N of Asn O-linked
glycan OH group Ser, Thr
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N-linked Glycosylation
Contains N-acetylglucosamine linked to amide
group of Asn N-linked motif Asn-X-Ser/Thr
where X represents any amino acid Common
pentasaccharide core (trimannosyl core)
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N-linked Glycosylation
N-linked complex glycan
Trimannosyl core
Asn
Asn
Mannose
N-acetylneuraminic acid
Galactose
N-acetylglucosamine
Fucose
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O-linked Glycosylation

• Contains N-acetylgalactosamine linked to
• hydroxyl group of Ser or Thr

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O-linked Glycosylation
Ser
N-acetylneuraminic acid
N-acetylgalactosamine
Galactose
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Filgrastim MW core protein 18.6 kDa
G-CSF MW glycoprotein approx. 20 kDa
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Recombinant G-CSF
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Erythropoietin (EPO) a clinically useful
glycoprotein
Primary regulator of erythropoiesis Gold
Standard for treating anemia associated with
renal failure 30 kDa protein ? 40 of proteins
mass is composed of carbohydrates
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Carbohydrate Structure of EPO Varies
Isoform (No. of Sialic Acid residues)
3 N-linked chains
1 O-linked chains
14 13 12 11 10 9
Br. J. Cancer (2001) 84 (Supplement 1), 3-10
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In vivo Efficacy of EPO Isoforms
Br. J. Cancer (2001) 84 (Supplement 1), 3-10
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Cell culture
Goal To produce a recombinant protein from a
living system (cell) To achieve this goal Must
be able to grow or culture these cells.
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Systems to express recombinant proteins
Bacteria Yeast Mammalian cells Also. Plants In
sect cells (baculoviruses) Transgenic animals
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Choice of Expression System
Growth pattern of cells (slow or fast) Cost of
growing cells Level of expression of the product
(high or low) Ease of product purification
Post-translational modifications Proper folding
and activity of protein product Availability of
suitable vectors
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Bacteria
E. coli most common strain Advantages Rapid
growth on low-cost media Easy to scale-up from
lab to production Disadvantages Proteins
produced in E. coli are not glycosylated. Express
ed protein may aggregate or fold improperly
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Yeast
Advantages Eukaryote Splices pre-mRNA
Glycosylates proteins Rapid growth on
low-cost media Secretes product into the media
more readily than E. coli Disadvantages Glycosy
lation pattern may be different than the parent
protein Expressed protein may aggregate or fold
improperly
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Mammalian Cells

• Chinese hamster ovary cells (CHO)
• Baby hamster kidney cells (BHK)
• Hybridomas used in monoclonal antibody production
  
• Advantages
• Post-translational modifications (glycoslyation
  and folding) more closely resemble human
• Properly splices pre-mRNA
• Disadvantages
• Much slower growth than bacteria or yeast.
  Doubling time 18-24 h vs. 20-90 min bacteria)
• More difficult to culture
• More expensive to culture

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Usually endotoxin or LPS of gram negative
bacteria
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Expression systems used to produce FDA approved
recombinant drugs
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Purification of recombinant proteins
1) Purify the recombinant drug from the myriad
of proteins in the producer cell. 2) Remove
potentially dangerous contaminants that may be
present in bacteria, mammalian cells or serum.
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Contaminants that may be present following
manufacturing

• Viruses
• Bacteria
• Pyrogens (e.g.,LPS)
• Cellular DNA
• Proteins

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Purification of recombinant proteins
Particulate removal
Concentration
Capture/Initial purification
Intermediate purification
Final purification
Sterilization/Formulation
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Purification of recombinant proteins by
chromatography
Chromatography column that is modified to
interact with our protein drug but not the other
proteins in the cell. Charge Specific
interactions
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It takes about two months to produce one lot of
Epotein alpha from starter inoculum to
ready-to-ship vial.
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Protein Purification Issues
N and C terminal heterogeneity formyl-met,
protease digestion Chemical modifications/conforma
tional changes splice variants, folding
intermediates deamidation, hydroxylation,
etc. Post translational modifications
glycosylation, phosphorylation, fatty acids,
etc. Proteolysis/degradation Inclusion body
formation
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Manufacturing of recombinant proteins from
cultured cells
Revolutionized the number and amount of drugs
that are available to treat disease. Complex and
expensive venture. Investment of 25 million
required to produce a modest amount of a protein
drug costs passed down.
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Transgenic technology as an alternative to
drug-producing cells
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Transgenic Technology

• Introduction of genes (including human genes)
  into the germ-line cells of plants and animals.
• Provides stable introduction of foreign genes at
  the embryonic level.
• Transformed organisms will pass along the new
  genes to their offspring.

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Why Transgenic Technology?

• Study human diseases using a transgenic animal
  model
• Use transgenic animals or plants to produce a
  desired product (e.g., drugs)

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Transgenic Technology
Insert gene of interest into the pronucleus of a
fertilized ova. Implant into pseudopregnant
mouse. Isolate DNA from each of the offspring to
determine which offspring carries the
transgene. Continuous matings of the transgenic
animals will produce a stable transgenic
line. Offspring carries the foreign DNA in all
of its cells.
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Green Fluorescent Protein (GFP)
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Normal mice
Transgenic mice with GFP
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Transgenic animals as drug factories
Desirable to have the protein drug secreted in an
easily retrievable manner. A recombinant drug,
secreted into the milk of the transgenic animal
could be produced in large quantities and easily
retrieved from the animal.
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How may mammary tissue be used to produce
recombinant protein?
Attach the promoter sequence of a major milk
protein upstream of the drug gene. Although this
foreign gene will be present in all of the cells
of the transgenic animal it will only be
expressed in the mammary tissue.
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Human Protein C
Blood protein. Functions to control blood
clotting. Some individuals have inborn
deficiency require exogenous Protein C.
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DNA fragment containing new hybrid gene
Human sequence for protein of interest (i.e.drug)
Male Pronucleus
Mouse promoter sequence for a milk protein
Female Pronucleus
Collection of pig embryos
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Genie The first genetically engineered animal
to produce a human protein drug (human protein
C) in her milk.
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Genie

• Produced sufficient quantities of human protein
  C.
• 1 g of human protein C per 1 liter of milk.
• 200-times more than present in human blood.
• About 1/3 of the rProtein was biologically
  active.

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Some examples of therapeutic protein production
using transgenic animals
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Cloning
Whole nucleus of any cell type is used. Progeny
are genetically identical to parent. All progeny
carry same genetic material.
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Process of Cloning
Parental cell
Ova
Nucleus removed and injected into enucleated
ova (nuclear transfer)
Discard nucleus
Enucleated ova (no nucleus)
Ova containing new nucleus A CLONE
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Animals Cloned (as of early 2003)
Sheep Cow Pig Goat Mouse Cat Gaur (endangered
species) Rat Wild sheep Banteng (endangered
species)
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Dolly - The First Cloned Sheep
Dolly was an identical genetic copy of her
mother. A technological breakthrough. From a
pharmaceutical perspective, not very
useful. However, if one could make a clone that
expressed a foreign gene, that would be
beneficial. - a transgenic clone
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Transgenic Animals by Cloning
Dolly - is a clone, not a transgenic animal
since she doesnt contain a transgene Polly -
transgenic sheep (nuclear transfer) contains
gene for human growth factor IX Major
differences between transgenics and cloned
animals is the amount of DNA used to generate
them. small piece of DNA (a gene) versus whole
nucleus
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Serum Albumin

• Major blood protein, regulates blood pressure.
• Annual world-wide demand, 400-500 metrics tons.
• Presence HIV, blood pathogens limits amount of
  albumin
• from blood.
• Estimated that a herd 2,000-3,000 cows could
  produce
• enough serum albumin to meet demands.

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Production of human serum-albumin in cows milk
by cloning

• Begin with a vector that contains the gene for
  human serum-albumin placed down-stream of a
  promoter for a milk protein (b-casein). Vector
  contains an antibiotic resistance gene (e.g,
  neomycin).
• Introduce vector into female, fetal cow cells.
• Select on neomycin. Vector will become
  integrated into chromosome of host cell.

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Create plasmid (vector) containing gene of
interest
Human serum albumin gene (HSA) downstream of
promoter for milk protein (b-casein)
HSA
p
Neomycin resistance gene (selectable marker)
Neo
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Introduce vector into female fetal cow cells
HSA
p
Neo
Select on neomycin. Vector will become
integrated into chromosome of host cell.
Neomycin resistant transduced cells
Neomycin kills non-transduced cells
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Plant transduced and selected ova into surrogate
mother
Ova containing transduced nucleus
Calf is a clone of the animal that supplied the
fetal cow cells. Human serum albumin will be
produced in the milk. All of the calves born will
be females and thus produce milk.
surrogate cow
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Cow cloning, contd
Take an ovum from a different cow and remove the
nucleus. Fuse nucleus of antibiotic resistant
cells with the enucleated ova. Plant into
surrogate mother. Calf is a clone of the animal
that supplied the fetal cow cells. Human serum
albumin will be produced in the milk. All of the
calves born will be females and thus produce
milk.
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Transgenic vs. Transgenic Clone
Transgenic -Laborious -Inefficient- Not all
injected eggs incorporate DNA with human
gene. -Expensive in large animals sheep, cows,
pigs. -Trial error breeding to obtain female
milk producers.
Cloning a transgenic - Use antibiotic resistance
marker to select for gene transfer, more
efficient. - Herd of drug producing animals
will be clones or 100 genetically identical.
-100 of clones will be female milk
producers.
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Definitions
Clone - Identical copy of parent made by nuclear
transfer all progeny are clones (the same)
Dolly Transgenic - Contains a foreign gene
not all progeny are transgenic Genie (human
protein C) Transgenic Clone - Copy of parent
plus contains a foreign gene all
progeny are transgenic HSA producing
cow