BIOTECHNOLOGY: THE EXPRESSION OF FOREIGN PROTEINS IN

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BIOTECHNOLOGY THE EXPRESSION OF FOREIGN
PROTEINS IN BACTERIA
COURSE FIGURES CAN BE DOWNLOADED THROUGH
http//www.courseweb.uottawa.ca/BIO4174
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BIOTECHNOLOGY Expression Systems
Background Issues
Why not just purify them, it has been done
before? Extract from tissues insulin from
pig pancreas growth hormone from cadaver
pituitary glands Extract from blood factor
VIII for hemophilia albumin for IV solutions
and media Extract from urine erythropoietin
(made in kidneys)
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BIOTECHNOLOGY Expression Systems
Background Issues

• Why are heterologous systems needed?
• Natural sources may be rare and expensive, hard
  to isolate
• and possibly contaminated with pathogens such as
• viruses (hepatitis, HIV) or prions (CJD, vBSE).
• Recombinant proteins may be more abundant,
• cheaper and safer.
• Individuals or groups may have objections to the
  use of
• animal or blood products.
• Use of the human protein minimizes immune
  reactions.

see notes
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BIOTECHNOLOGY Expression Systems
Background Issues

• What the general issues to cloning in a
  heterologous host?
• Selection of host and vector
• Can regulate expression by choice of vector
• Genetic modification of host and cloned gene
• Choice of location of product
• Modification of protein produced
• Ease of production and scale
• Can facilitate purification

Each of these parameters can be modified in each
host
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BIOTECHNOLOGY Expression Systems
Background Issues
The Production Of Recombinant Proteins Each
System Has Advantages And Disadvantages
Q What perspective is missing from this chart?
Ma et al. (2003) Nature Reviews Genetics
4794-805.
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BIOTECHNOLOGY Expression Systems
Background Issues

• Many factors may influence our choice of
  vector/host.
• Compatibility between vector and host.
• Availability of versatile vectors, regeneration
  systems etc.
• Regulatory environment and FTA(IP).
• Costs
• The complexities of altering metabolism!!

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BIOTECHNOLOGY Expression Systems
Background Issues

• Design Problems and Solutions
• Availability of strong, regulated promoters and
  other signals (RBS, termination signals,
  processing signals, RNA stability).
• Improvements to mRNA (stability, remove cryptic
  splice sites, etc.)
• Codon optimization for heterologous expression.
  Different organisms show codon bias and
  switching hosts presents a problem.

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BIOTECHNOLOGY Expression Systems
Background Issues
For codon optimization, GenScript
bioinformaticians take many other factors into
consideration, e.g. secondary structure, GC
content, repetitive codon etc., and developed a
proprietary algorithm. GenScript algorithm can
optimize sequences for protein expression using
either your own codon usage table or those from
publicly available codon usage database. It can
also converts your amino acid sequence into a DNA
sequence with overall codon usage similar to a
specified organism, and also optimizes the RNA
secondary structure. http//www.genscript.com/in
dex.html
You should be aware that codon usage CAN affect
protein function! See Kimchi-Sarfaty et al.
(2007) A "Silent" Polymorphism in the MDR1 Gene
Changes Substrate Specificity Science 315 525 -
528.
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BIOTECHNOLOGY Expression Bacteria
There are many systems for expression. Some
commonly used ones
Course Map Plasmid Vectors Viral
Vectors Specialized Vectors Escherichia coli
Other Bacteria Yeast Pichia pastoris
Baculovirus Cell Culture Plants Animals

• Escherichia coli
• Other bacteria
• Yeast
• Pichia pastoris
• Baculovirus
• Animal cell culture
• Plants
• Animals (sheep, cows, goats)

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BIOTECHNOLOGY Expression Bacteria

• Escherichia coli has been the " factory" of
  choice!!
• A single-celled organism, it reproduces
  asexually (genetics)
• It is easy and cheap to grow on simple food
  sources.
• It has rapid growth and high yield (50-500mg/L)
• It can be easily engineered (you in the BIO/BCH
  labs).
• It has a tremendous range of vectors and genetic
  resources,
• including promoters and regulatory systems.
• We know a lot about it (safety) and what it does
  in culture.

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BIOTECHNOLOGY Expression Bacteria

• But E.coli is not without potential problems for
  the cloning of
• foreign proteins.
• Need to make fusions with host sequences such as
  promoters
• As with all prokaryotes, it lacks membrane bound
  organelles.
• Post-translational modifications such as protein
  processing and
• glycosylation are usually absent.
• As with all prokaryotes, it lacks (or may lack)
  pathways for proper
• folding or interaction with co-factors.
• Problems with large (gt50kD) or S-S rich proteins
• May not be useful for complex reactions with
  co-factors.

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BIOTECHNOLOGY Expression Bacteria
A simple example Cloning of insulin
Background Information Human insulin
can be produced in bacteria as it has no
post-translational modifications and it a small
protein. Insulin is produced in pancreatic cells
as preproinsulin (1) an N-terminal signal
sequence of 16 amino acids, (2) a B-chain of 30
amino acids, (3) a C-chain of 33 amino acids,
and (4) an A-chain of 21 amino acids The
N-terminal and the C-peptides are cleaved, and
disulfide bonds are formed between the A-chain
and the B-chain.
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BIOTECHNOLOGY Expression Bacteria
Original cloning strategy for the production of
insulin by Genentech

• The vector
• what is its role?
• what factors influence our choice?
• how do the choices affect the cloning outcome?
• The insert
• what is the source of the gene?
• what is a cassette?
• where does the regulatory DNA originate?
• how do we optimize expression ?
• The host
• how does the host influence expression of the
  gene?
• how does the host influence choice of vector and
  insert?
• eucaryotic hosts are more complex!?!

Experimental 1. Correct DNA sequence. 2. RIA for
both chains. 3. Correct protein sequence. 4. By
EM, inclusion bodies in E. coli with protein
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BIOTECHNOLOGY Expression Bacteria
In E. coli, foreign proteins expressed at high
levels may not fold correctly and are recovered
as inclusion bodies.
Refold in vitro /- any cofactors. OR May not
need to refold.
Examples of inclusion bodies
http//www.proteomtech-inc.com/opPrinciple.html
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BIOTECHNOLOGY Expression Bacteria
Commercial Production ( E. coli)
Name/Product Market Replaced Humulin
(Human r-Insulin) Type I diabetes Animal
insulin Somatropin (Human growth hormone)
Dwarfism Pituitary Actimmune (interferon
gamma-1b) Immunomodulator New Pulmozyme
(DNAse) Cystic Fibrosis New Retavase(tissue
plasminogen activator) Heart attack New http//b
iobasics.gc.ca/english/view.asp?x556
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BIOTECHNOLOGY Expression Bacteria
Improved vector design allows improved
purification protocol
All of these proteins were expressed in E. coli
as GST fusions, the proteins run on an SDS gel,
and the western blot was probed with an anti-GST
antibody. Many of these proteins were tested and
found to be functional in standard assays. Why
use this system (or a similar one) and how does
it work? (the dot indicates the expected size)
Braun, R. et a. (2002) Proc Natl Acad Sci 2002
March 5 99(5) 2654-2659. Proteome-scale
purification of human proteins from bacteria.
http//www.ncbi.nlm.nih.gov/pmc/articles/PMC12240
3/
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BIOTECHNOLOGY VECTORS
Vectors for protein purification
pGEX Fusion Vectors HOW ARE THEY USED??
WHAT ARE POTENTIAL PROBLEMS WITH THIS VECTOR??
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BIOTECHNOLOGY VECTORS
Vectors for protein purification
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BIOTECHNOLOGY Expression Bacteria
The previous two examples are only a small
fraction of the 1000s of publications where a
single protein is expressed in bacteria, mainly
E. coli! Two recent examples illustrate a very
promising approach-cloning metabolic pathways
into E. coli for the production of the proteins
or reagents via complex reactions! We will
discuss the progression of Artemisinin expression
as an example.
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BIOTECHNOLOGY Expression Bacteria
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BIOTECHNOLOGY Expression Bacteria
Background Artemisinin is a promising
next-generation anti-malarial drug that is badly
needed in African and South American countries
where the disease is becoming resistant to
cheaper front-line medications. But the cost of
extracting, purifying and concentrating the
active ingredient from the wormwood plant
(right) is often prohibitive. We've reconstituted
a metabolic pathway from yeast in E. coli,
synthesized the drug precursor gene from
wormwood, then optimized the pathway for
10,000-fold increase in productivity, to perform
simply and cheaply many of the chemical steps
needed to synthesize the drug
Most of our medications are small,
non-proteinaceous materials originally found in
plants as so-called secondary metabolites. As
the name implies, they are not made by metabolic
pathways that are conserved in bacteria. You can
grow a lot of plants and extract the compound.
But can we make enough or can we make them in
bacteria? And in bacteria you can do engineering!
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BIOTECHNOLOGY Expression Bacteria

• Codon optimization (ADS, amorphadiene synthase )
• Use of E. coli mutations
• Many yeast genes, algal gene
• Black triangles represent the PLAC promoter and
  tHMGR refers to an N-terminal truncated product
  of the native HMGR gene. Gene symbols and the
  enzymes they encode (all genes were isolated from
  S. cerevisiae except where noted)
• atoB, acetoacetyl-CoA thiolase from E. coli
• HMGS, HMG-CoA synthase tHMGR, truncated HMG-CoA
  reductase
• ERG1, mevalonate kinase
• ERG8, phosphomevalonate kinase
• MVD1, mevalonate pyrophosphate decarboxylase
• idi, IPP isomerase from E. coli
• ippHp, IPP isomerase from Haematococcus pluvialis
  (alga, member of Chlamydomonadales)
• dxs, 1-deoxy-D-xylulose 5-phosphate synthase
• ispC, 1-deoxy-D-xylulose 5-phosphate
  reductoisomerase
• ispA, FPP synthase from E. coli
• ADS, amorphadiene synthase.

Q Where do you find these genes and sequences?
Martin et al. (2003) Engineering a mevalonate
pathway in Escherichia coli for production of
terpenoids. Nature Biotechnology 21 796 - 802.

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BIOTECHNOLOGY Expression Bacteria

• How can you improve the production of
  artemisinin?
• Further metabolic engineering of vector/host
• Try a different vector/host system

• Steps taken by R-J et al.
• (boring but necessary RD)
• Identify rate-limiting step(s)
• Modify enzymes (codon bias,
• promoters etc.)
• Change replicons etc.
• Carefully monitor all changes.

Data from Redding-Johanson, TS, et al. (2011).
Targeted proteomics for metabolic pathway
optimization application to terpene
production. Metab. Eng. 13194-203
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BIOTECHNOLOGY Expression Bacteria
How can you improve the production of
artemisinin? Further metabolic engineering of
host Try a different vector/host system

Data from Ro, D.K. et al. (2006) Production of
the antimalarial drug precursor artemisinic acid
in engineered yeast. Nature 440 940-943. Also
Farhi, M. et al. (2011) Generation of the potent
anti-malarial drug artemisinin in tobacco. Nature
Biotechnology 29 10721074.
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BIOTECHNOLOGY Expression Bacteria (update, see
Notes)
Comparing E.coli and Yeast E.
coli Yeast Recovery 22-112 mg/L
153mg/L Location Intracellular? Excreted and
bound Purification Not attempted Ether
extract/silica column To optimize would need
metabolic engineering! Caveat While this may
solve the production problem, does it solve the
distribution and identity problems?

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BIOTECHNOLOGY Expression Bacteria
Other potential bioproducts 1. Terpenoids
(isoprenoids) Over 40,000 isoprenoids are
known, including TaxolTM and artemisinin. Others
fight viral infections and cancers. Commonly
extracted flavors, fragrances and
neutraceuticals. 2. Degradation of pollutants,
antibiotic synthesis. 3. Produce your own
alkaloids in yeast (2008). 4. Use you
imagination and think BIG!!!

This approach has lead to many examples of
synthetic biology
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BIOTECHNOLOGY Expression Bacteria
Other potential bioproducts-Biofuel

LS9 has created industrial microbes that
efficiently convert renewable feedstocks to a
portfolio of... hydrocarbon-based fuels and
chemicals. LS9's unique technology provides a
means to genetically control the structure and
function of its fuels... ...efficiently
converting fatty acid intermediates into
petroleum replacement products via fermentation
of renewable sugars LS9 has also discovered and
engineered a new class of enzymes and their
associated genes to efficiently convert fatty
acids into hydrocarbons.
Questions Is making fuel from corn desirable? Is
making fuel from waste desirable? What is waste?
See 1. Zhang et al. (2012) Design of a dynamic
sensor-regulator system for production of
chemicals and fuels derived from fatty acids.
Nature Biotechnology 30 354359. 2. Algae
http//www.joulebio.com/
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BIOTECHNOLOGY Expression Bacteria

• Escherichia coli summary
• Great host (see slide 11).
• It can be easily engineered for the production
  of proteins
• and simple compounds.
• Can do metabolic engineering. We described
  artimisinin and
• fuels but has been done for others. e.g.
  taxol-see
• P. K. Ajikumar et al., Science 330, 70 (2010).
• It is an easily scalable system for industrial
  purposes,
• Many cloning options. You have great flexibility
  in your choice
• of systems and host based upon extensive
  research.

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BIOTECHNOLOGY Expression Bacteria
Escherichia coli summary There are options to
overcome some of the disadvantages. Problem Solu
tions? 1. Codon bias Engineer gene or use host
such as BL21-CodonPlus-RIL for AT-rich genes
(extra copies of E. coli tRNA genes
argU, ileY, leuW (or BL21-CodonPlus-RP for
GC-rich genes) 2. Protein is toxic Use tight
promoter/regulator system use batch culture 3.
Protein is unstable Express as fusion use
protease negative host 4. Poor
expression Titrate reagents each time for growth
and induction export protein or use
inclusion body approach engineer host
to overcome substrate limitations. 5. If the
above fail switch hosts e.g. to a yeast system.
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BIOTECHNOLOGY Expression Bacteria

• Expression in Bacteria
• Simple systems
• Very competitive to market leading to market
  replacement.
• Fewer barriers to market entry.
• Range of bacteria can be used, not only E. coli.
• Can apply the same ideas to other single cell
  systems
• including yeast, fungi and algae.
• Potential for genetic improvement
• but only for certain proteins!

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BIOTECHNOLOGY Expression Bacteria
Relationship of the section Expression Bacteria
to the NSERC projects
Excellent host for expression and cloning Can
use to assemble constructs and test
expression Can be useful for directed
improvement of function Where do you go to find
genes and regulatory sequences? Consider cloning
approach and then metabolic engineering