CellFree Expression of Proteins for NMR

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Cell-Free Expression of Proteins for NMR
Dave Aceti
11/12/99
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Pros and Cons of Cell-free Expression

Pro
Con

• Incorporation of labeled and/or
• unnatural amino acids
• site-specific
• specific for residue type
• specific for portion of protein
• Other methods (in vivo, synthetic, semi-
• synthetic, chemical modification) are
• less specific and/or limited by protein size.
• May prevent inclusion body formation
• May prevent proteolysis
• Cyto-toxic proteins can be expressed

• Yields generally low (lt 100 ug/ml)
• (OK for verification of gene product)
• Some components expensive
• RNAase-sensitive

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How Cell-Free Expression Works
Major Components
Template Promotor/Gene-to-be- expressed. May be
circular/supercoiled or linear DNA (depending on
promotor).
GENE
T7 p
Substrates NTPs, Amino Acids, ATP
Enzymes/Reusable Factors T7 RNA Pol., tRNAs,
EFs, ribosomes, ARS
ATP Replenishing System creatine kinase,
creatine phosphate
Inhibitors RNAase Inhibitor, NaN3
Supplied by E. coli extract
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A Short History

• 1960s - First in vitro protein synthesis
  (Niremberg and Matthaei)
• E. coli extract system
• Used in analysis of genetic code

• Innovations increased yield (G. Zubay)
• Degraded endogenous DNA/RNA in E. coli extract
• Optimized components
• Coupled transcription and translation

1973 -

• 1970s - Eukaryotic systems developed, e.g.,
• wheat germ
• rabbit reticulocyte

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A Short History
1988, 1991 - Continuous flow system increases
reaction time, yield (Spirin et al., Kigawa
Yokoyama)
1989 - Site-specific incorporation of unnatural
amino acids using suppressor tRNA (Schultz)

1996 - Semi-continuous flow (dialysis) method
(Kim and Choi, Davis et al)

• 1999 - Yield increased to mg/ml range (Kigawa et.
  al)
• improved energy regeneration system
• condensed E. coli extract
• optimized other components

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Semi-continuous flow reaction unit (Davis et al.,
Promega Notes)
10-50 kDa MWCO
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(No Transcript)
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Templates
Used in Reactions
Planned
Protein Encoded
Plasmid Template
Ferredoxin, Human pET3a-HuFd X Ferredoxin,
veg. pET9a-VgFd X Brazzein
pET3a-SW X Brazzein pET9a-SW X Synaptotagmin
, C2A pET9a-C2A X Lymphotactin, Human
pET9a-HuLn X Phosphoglucomutase pET3a-Pgm X
Ovomucoid, 3rd domain X Myelin Basic
Protein X
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Results
External Solutions
Reaction
________________
_______________
HuFd
HuLn
Pgm
HuFd
HuLn
Pgm
SW
SW
SM (kDa)
66
55
Reaction Set 1 pET3a-SW pET9a-HuLn pET3a-Pgm pET9
a-C2A
37
31
21
14
Reaction Set 2 Protein pET9a-SW 6.4
kDa pET9a-HuLn 11 kDa pET3a-Pgm 63
kDa pET3a-HuFd 13.7 kDa Changes 1)
ferredoxin template 2) Tyrosine solubility 3)
3-5 cAMP solubility
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Future Work
1. Improve template purity 2. Re-make
solutions 3. Re-make E. coli extract 4. Improve
RNAse inhibition 5. Vary Mg2 and PEG 8000
concentrations 6. With success Experiment with
labeling patterns
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Incorporation of An Unnatural Amino Acidat a
Single Site