Expressing UDPGlycoslytransferases from Medicago truncatula in the yeast Pichia pastoris

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Expressing UDP-Glycoslytransferases from
Medicago truncatula in the yeastPichia pastoris
Trilby Hillenbrand Wang Lab
Asaf Madi et al, Agricultural Research
Organization of Israel, 2006
Xiaoqiang Wang, Protein Structures and Function
presentation, Noble Foundation 2006
Thomas Mainczyk et al, Technischen Universitat
Berlin, 2003
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Glycosyltransferases

• Transfer of sugar moiety from an activated donor
  substrate to an acceptor molecule
• Most proteins, lipids and natural products are
  glycosylated
• Facilitates bioactivity and storage of molecules
• Over 80 glycosyltransferase families

Wang, Protein Structures and Function
presentation 2006
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UDP-Glycosyltransferases (UGTs)

• Belong to Family 1 of Glycosyltransferases
• Have been identified by PSPG motif
• 27 UGTs identified in human genome
• Over 100 predicted for Medicago truncatula
• Involved in the synthesis of natural products

Shao et al, 2005
Wang, Protein Structures and Function
presentation, 2006
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Structure of UGTs

• Determine structure of UGTs
• Better understand function
• Manipulation of UGTs and increased production of
  secondary metabolites

Xiaoqiang Wang, Protein Structures and Function
presentation, Noble Foundation 2006
Wang et al, Structural Biology Lab, 2006
Steve Hughes, Genetic Resource Center, Adelaide,
Australia
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Expression of UGTs

• Success of E. coli
• mtUGTs 22D, 49F and 83F among others
• Others have not yet succeeded in E. coli
• mtUGTs 12E, 56E and 68E among others

Escamilla and Wang, Summer Project Proposal 2006
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Pichia pastoris An alternative expression system

• Benefits of alternate expression system
• Higher expression levels than Saccharomyces
• Utilize most of the posttranslational
  modifications of higher eukaryotes
• Easy and inexpensive

Invitrogen, Easy Select Pichia Expression Kit
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Objective

• Test Pichia pastoris as an alternative system for
  expressing UGTs from Medicago truncatula

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Project Overview
Cloning into UGT-TOPO vector
Sequence
PCR target DNA
Target DNA
Protein Expression
Sequence
Transform P. pastoris
Subcloning into pPICZB vector
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Cloning into TOPO Vector
12E 22D 56E
UGT-PET28 Vector (Target DNA)
PCR
UGT Fragment
Transform in E. coli
Zeocin
Kanamycin
Zeocin
Kanamycin
UGT-TOPO Vector
PCR-Blunt II-TOPO
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Cloning into TOPO Vector

• Analyzing Colonies

1 2 3 4 5 6 7 8 9
Minipreps
1 2 3 4 5
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Subcloning into pPICZB
UGT Fragment
Purify UGT fragment from gel
Xba1 BstB1
UGT-TOPO
Purify Vector fragment from gel
Xba1 BstB1
pPICZB Fragment
pPICZB
UGT Fragment
Transform in E. coli
Ligase
pPICZB Fragment
UGT-pPICZB
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Subcloning into pPICZB
12E 22D 56E pPICZB
Gel Purification (QIAquick Gel Extraction Kit
and GFX DNA and Gel Band Purification Kit)
Ligation
Transformation
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Subcloning into pPICZB
Analyzing Colonies
Minipreps
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Transformation into Pichia pastoris
Middle Scale DNA preparation
Linearize UGT-pPICZB
Transform P. pastoris
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Future Directions

• Express and purify protein
• Check the activity using a pool of substrates
• Produce large scale to crystallize and determine
  structure
• Pichia pastoris clones can be tested for
  biocatalysis using whole yeast cells

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Acknowledgements

• Dr. Luis L. Escamilla-Trevino
• Dr. Xiaoqiang Wang and Lab
• Steve Rhines and Emily Edwards
• Samuel Roberts Noble Foundation
• Kirsti Burr, Emily Combs and Feng Zhang