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CellFree Expression of Proteins for NMR

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E. coli extract system. Used in analysis of genetic code ... Degraded endogenous DNA/RNA in E. coli extract. Optimized components ... – PowerPoint PPT presentation

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Title: CellFree Expression of Proteins for NMR


1
Cell-Free Expression of Proteins for NMR
Dave Aceti
11/12/99
2
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

3
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
4
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

5
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

6

Semi-continuous flow reaction unit (Davis et al.,
Promega Notes)
10-50 kDa MWCO
7
(No Transcript)
8
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
9
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
6
10
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
11
Incorporation of An Unnatural Amino Acidat a
Single Site
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