Download Protein Expression Issues in Protein Expression

Protein Expression
Problems, Problems, Problems
Issues in Protein Expression
• Two types of expression
– Homologous
– Heterologous
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Homologous Expression
• Requires expression vector
• Transformation system
– Systems available
•
•
•
•
Yeast
Insect -- Sf9 cells
Fungal -- Several species
Mammalian
– Chinese Hamster Ovary cells
– HEK 293 Cells
• In many cases transformation is difficult
Heterologous Expression Problems
• Toxic proteins
– Can use tight regulation of expression
• Missing modifications
– Addition of cofactors, metals, etc.
• Missing interactions
– Requires other proteins in complex to fold
properly
• Missing tRNA
– Codon usage varies between organisms
and kingodms
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Heterologous Expression
Problems
• Expressed proteins are
improperly folded or mixed with
other proteins
– Inclusion bodies
– Can be >= 90% of expressed
protein.
– Protein is improperly folded and
mixed with other proteins.
– Requires purification and refolding.
– Refolding is only partially
successful
http://web.mit.edu/king-lab/www/research/Scott/Scott-Research.html
Protein Microarrays
What do you want to know?
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Types of arrays
AM
Capture labeled haptens
Sandwich ELISA
FM
FM
AM
RP
Purified recombinant
proteins for P-P
interactions or substrate
--AM = Analytical micro array
--FM = functional micro array
--RP = Reverse Phase
micro array
Purified recombinant
proteins to detect Abs
Reverse phase
M. Walther, B. Stillman, A. Friße and J Beator,
Fast guide to protein microarrays, Whatman
What is necessary to make an
array?
• What is necessary to make an array?
– 1 or 2 specific antibodies for each protein
– All proteins to be studied are purified.
– A support and a detector
• What formats are available.
– Glass slide “chips”
– Nano-wells
– Nitrocellulose membranes
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Antibody Production
• Inject purified protein into mice and
produce monoclonal antibodies one at a
time.
• Inject organism or mixture into mice and
produce multiple monoclonal antibodies.
• In vitro antibody generation
– Phage display
– Expression library
Expression Library
Cells encoding
Ig genes
cDNA synthesis
IgM and IgA producing cell lines
cDNA
Amplification of complementarity determining region (CDR)
Pool of diverse DCRs
Framework oligonucleotides
E Söderlind, L Strandberg, P Jirholt, N. Kobayashi, V.Alexeiva, A-M Åberg, A Nilsson, Bo Jansson, M Ohlin, C Wingren, L Danielsson, R
Carlsson, and C A.K. Borrebaeck 2000 Recombining germline-derived CDR sequences for creating diverse single framework antibody libraries
Nature Biotechnology 18:852-856
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Expression Library
Framework oligonucleotides
Pool of diverse DCRs
Combination of oligonucleotides. Overlap extension gene synthesis.
linker
scFv antibody fragment gene
Antibody limitations
• Specificity
• Binding strength
• Production
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Genomic Protein Expression
• Major recombinational cloning strategies
– Gateway recombinational cloning system
– Gap repair-mediated recombination.
– Ligation independent cloning
Genomic Cloning Strategies
att B1
X
X Mix
T4 pol
+dC
att B2
Rxn 1
att P1
att P2
att L1
att L2
T4 pol
+dG
A
B
Mix
C
X
att R1
Rxn 2
att R2
A - Gap repair-mediated recombination
B - Ligation independent cloning
C - Gateway recombinational cloning system
E Phizicky, P. I. H. Bastiaens, H Zhu, M
Snyder & S Fields 2003 Protein analysis on
a proteomic scale Nature 422:208-215
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λ Phage insertion/ recombination
•
•
When the λ phage with att P site
inserts into genome att B site it
creates att L and att R sites.
Excision of the phage results in the
regeneration of the att B and att P
sites.
att P
att B
insertion
att L
att R
excision
att B
att P
Purification of Expressed Proteins
• Purification on a genomic scale requires
an rapid and specific purification process.
• Typically used are
– Affinity tags
– Tandom Affinity Purification (TAP)
• Double tagging
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Affinity Tags
– glutathione S-transferase
• Binds to glutathione coupled
column
• Removed with glutatione
– hexyl histidine
• Binds to Ni+ chelating column
• Removed by addition of
imidazole
– calmodulin binding protein
• Binds to calmodulin coupled
column
– protein A
• Binds to antibody column
• Removed by denaturation
– maltose binding protein
• Binds to Maltose coupled
column
• Removed with maltose
– FLAG
• Binds to FLAG antibody
column
• Removed by denaturation
– Strep II tag
• Binds to Avidin coupled
column
• Can be removed with
desthiobiotin
Expression systems
• Expression systems
– E. coli
– Insect cell line Sf9
– Yeast (Pichia pastoris)
– Other cell lines.
• Issues
– RNA processing (alternative splicing)
– Protein processing (modifications)
• Phosphorylation, cofactors, etc.
– Solubility
– Toxicity
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Attachment of proteins
• Want proteins to
– Maintain structure and functionality
• Includes keeping access to functional site
– Achieve maximal binding density
• For some applications nanowells are
needed.
Attachment of proteins
• Different surfaces
– Random attachments
• Covalent: amines, aldehyde- and epoxyderivatized glass surfaces
• Non-covalent: nitrocellulose, gel pads, or poly-Llysine
• Affinity tagged surfaces (oriented)
– Nickel
– Anti tag antibodies
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Detection
• small molecule probes
– fluorescent, affinity, photochemical, or
radioisotope tags
– Uses common DNA array devices for
detection
• label-free detection
– Surface plasmon resonance (SPR)
• Detects changes in index of refraction
• Sensitivity 1 nM (~100 fold less than fluorescence)
– carbon nanotubes and nanowires (conductance)
– Micro-electromechanical systems cantilevers
Protein Microarray Uses
• Interactions/reactions
– Protein-protein
– Protein-DNA/RNA
– Protein-lipid
– Protein-drug
• Disease protein markers
• Protein substrate detection
– Phosphorylation
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