PCR Amplification of Rhizobium Purine Biosynthetic Genes and Cloning into an Expression Vector

1
PCR Amplification of Rhizobium Purine
Biosynthetic Genes and Cloning into an Expression
Vector

• Rachel M. Lawton
• Kimberly A. Mistiszyn
• Jeffrey D. Newman
• Lycoming College
• April 10, 1999

2
The Purine Biosynthetic Pathway
TPP
purF purD purN purL(QY) purM
PRPP
AIR
FGAR
FGAM
purEK purC purB
AICAR
GMP
purH purH
guaA guaB
IMP
purB purA
AMP
3
FGAR Amidotransferase Organization
glutamine
amide transfer
ATP-binding, cleavage
Type I
Single subunit
PurL
- 1295 aa (E.coli)
B, g - proteobacteria, eukaryotes

Type II
PurL
- 742 aa (B.subtilis )
Multi-subunit
All other prokaryotes
4
pCOS110 Map

• Initial partial sequencing out from Tn5
  identified purY, purQ, and purL mutations.
• Subsequent complete sequencing identified purC
  and YJII.

EEcoRI
1 kb
BBamHI
E
E
B
E
E
B
purL
purY
purC
purQ
YJII
Sequenced regions
5
IMPACT T7 Strategy
Choose Expression system
Express fusion construct
Clone target gene
Purify target protein
Analyze fusion construct
6
Why use IMPACT T7?

• From New England Biolabs
• Target gene fused to
• chitin binding domain allows affinity
  chromatography
• intein - self-splicing protein
• Rapid and simple purification of native protein,
  without an affinity tag
• Release of target protein from the fusion partner
  without proteases
• Chitin is stable, abundant, inexpensive, and
  reusable

7
IMPACT T7 Strategy
Choose Expression system
Express fusion construct
Clone target gene
Purify target protein
Analyze fusion construct
8
Primers

• NdeI and EcoRV restriction endonuclease sites
  were incorporated for start and stop primers.
• TAA, a stop codon, was not incorporated since it
  would prevent our ultimate goal of creating a
  fusion protein.

9
Polymerase Chain Reaction

• Rhizobium etli purY and purQ genes were
  amplified by Polymerase Chain Reaction
• PCR product was purified using QIAquick
  Purification Membranes

10

Multiple Cloning Site (MCS)

• Want to incorporate PCR product into NdeI and
  SmaI restriction sites of pTyb2 vector

11
Restriction Sites
pTyb2

• Did not incorporate SmaI as stop site in PCR
  product due to the CCC codon, which codes for
  Proline
• EcoRV still gave blunt end, and sequence codes
  for Aspartate instead of Proline

NdeI
SmaI
NdeI
EcoRV
PCR product
12
Restriction Enzyme Digests

• pTyb2 vector cut with both NdeI and SmaI
• Ligation
• Transformation of E. coli
• Plasmid minipreps
• Restriction digests (looking for possible clones)

13
Restriction Enzyme Digests

• pTyb2 vector cut with both NdeI and SmaI
• Ligation
• Transformation of E. coli
• Patched colonies
• CTAB minipreps
• Restriction digests (looking for possible clones)

14
Clones!!!!

• Confirmed clones by PCR.
• Transformed into ER2566, an E.coli strain for
  expression studies

15
IMPACT T7 Strategy
Choose Expression system
Express fusion construct
Clone target gene
Purify target protein
Analyze fusion construct
16
Expression of the Fusion Protein

• IPTG induction of T7 polymerase
• T7 polymerase drives expression of fusion protein
  from T7 promoter.
• Cell extracts were analyzed by SDS-PAGE
• purQ fusion protein (80 kDa) apparent
• purY fusion protein (65 kDa) not produced

17

Why no R.etli purY fusion protein?

• Sequence analysis revealed that PCR product was
  cloned into SmaI site (i.e. vector had not been
  cut with NdeI).
• Other clones are currently being analyzed.

18
Conclusions

• R.etli purQ gene successfully cloned andfusion
  protein expressed.
• Several R.etli purY clonesbeing analyzed for
  correctcloning junctions.
• The project continues
• S. aureus, R. etli Type II FGAR amidotransferase
• E. coli, H. sapiens Type I FGAR amidotransferase