Structural Genomics of Parasitic Protozoans (Trypanosoma, Leishmania) (SGPP East)

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Structural Genomics of Parasitic
Protozoans(Trypanosoma, Leishmania)(SGPP East)

• Protein Expression at the University of
  Rochester
• Mark Dumont, Elizabeth Grayhack. Eric Phizicky

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Ligation-Independent Cloning(Uses PCR-vector
overlap sequences missing one type of nucleotide)
Restriction sites
ORF
Digest
PCR, purify
Mix ORF and vector ( 5 min. no ligase) Transform
E. coli
T4 DNA pol. 3exo (i.e.) dGTP
T4 DNA pol. 3exo (i.e.) dCTP
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Technical issues with LIC

• 1. Tm of overhangs
• 2. Removal of dNTPs from PCR products
• 3. Complete cleavage of vector

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Advantages of Ligation Independent Cloning

• 1. Speed 1 hr T4 polymerase treatment and 5
  min annealing
• 2. Low Background Reported to be lt1
• 3. Directional
• 4. Minimal introduction of extra amino acids in
  fusions
• Requires a run of 14 bases lacking one of
  the four nucleotides at each end
• 5. Only requires short PCR primers
• (ORF-specific sequences 14 nucleotides)
• 6. Low expense Requires only T4 DNA polymerase,
  no
• proprietary reagents.

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Ligation Independent Cloning

• Selected References
• Aslanidis C, de Jong PJ, Schmitz G (1994) Minimal
  length requirement of the single-stranded tails
  for ligation-independent cloning (LIC) of PCR
  products. PCR Methods Appl. 4172-7.
• Haun RS, Serventi IM, Moss J. (1992) Rapid,
  reliable ligation-independent cloning of PCR
  products using modified plasmid vectors.
  Biotechniques 13515-8.
• Aslanidis C, de Jong PJ (1990) Ligation-independen
  t cloning of PCR products (LIC-PCR). Nucleic
  Acids Res.186069-74.
• Novagen Product Information

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Expression Vectors for Protozoan Soluble Protein
Expression

• Features
• Ligation-Independent Cloning-compatible
• N-terminal non-cleavable His6 in all vectors

PmlI
NdeI
Pml1

NdeI
ATG-His6-ATG......TAA
T7 terminator
Ribosome binding site
ORF
T7 promoter
PmlI
NdeI
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Expression of a test gene following
ligation-independent cloning and transformation
into BL21-DE3
(Coomassie stain)
Vector Insert (Independent isolates)
TPT1- 8 ?g
Vector
Tpt1p
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Pichia Pastoris for High Throughput Membrane
Protein Expression
1. Lower eukaryote- post-translational
modifications 2. Rapid cloning 3.
Inexpensive 4. High density cultures leading to
high yields per liter 5. Previous history for
membrane protein expression
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Cloning Strategy for Membrane Protein Expression

• Use ligation independent cloning to insert a
  single PCR-product into two E. coli vectors and
  two Pichia vectors

Single PCR product
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Expression Vectors for Protozoan Membrane Protein
Expression

• Current Features
• 1. C-terminal non-cleavable His6 ( c-myc in
  Pichia)
• 2. Vectors with and without cleavable N-terminal
  secretion signals
• pre-pro-a-factor followed by kex2p
  cleavage site in Pichia
• pelB followed by signal peptidase site
  in E. coli
• 3. Favorable translation context in Pichia.
  Optimal ribosome binding site in E. coli
• 4. Codons of fusion regions optimized for
  Pichia and E. coli
• 5. Zeocin selection for Pichia vector
• selectable in E. coli and yeast, allows
  selection for multicopy integrants in Pichia.
• Future
• 1. Multiple Affinity Tags, protease sites
  (rhinovirus 3C (Prescision))
• 2. Fluorescent expression screening

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Pichia LIC vectors with C-terminal Tags (Derived
from pPicZa from Invitrogen)

• Linearize for LIC by cutting at two BsmB1 sites
  (cuts outside recognition site)
• Additional PmlI site between BsmB1 sites to
  reduce background due to uncut vector
• Linearize at BglII and BamHI for integration
  into Pichia

a
without signal peptide
with pre-pro-a factor signal peptide
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• Technical Assistance
• Wayne Bowers
• Nadia Fedoriw
• Craig Menges
• Pichia consultant
• Ina Urbatsch

Wim Hol
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E. coli LIC vectors with C-terminal Tags
(Derived from pET22b from Novagen)

• Linearize by cutting at two sites with Bsm1
• (cuts outside recognition site)
• Additional PmlI site between BsmI sites to
• reduce background due to uncut vector

with pelB signal peptide
without signal peptide