Lab Director Fei Philip Gao, Ph.D. - hbc.ku.edu
Transcript of Lab Director Fei Philip Gao, Ph.D. - hbc.ku.edu
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For more information and lab coupons : https://psf.cobre.ku.edu/cores/ppg/aboutor search for “Protein Production” at KU main web page.
Lab DirectorFei Philip Gao, Ph.D.
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INTRODUCTION
The Protein Production Group (PPG) focuses on the cloning, expression and purification of prokaryotic and eukaryotic proteins for investigators in Kansas and the region.
The laboratory maintains a variety of equipment to support the production of properly folded proteins in quantities suitable for structural studies (X-ray and NMR), functional studies (catalytic or biological), label-free binding studies (SPR) and/or high throughput (HTP) screening studies.
It is a place where you will get a project done from a sequence on a piece of paper to a bucket of protein in hands.
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Work Request
• Gene to protein protocols• Cost estimate, Quotation #• Sign project number• Project waiting list
Protein Production
• Ordering supplies• Research workflow• Recording data and results
Deliverable• Samples• Scientific
report• Invoices
PPG OPERATIONAL OVERVIEW
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SamplesProtocols
PCR, LIC cloning, transformation
O/N culture, PCR check insert
DNA mini-prep, packing for sequencing,
transformation into Bl21(DE3)pRARE
Sequencing analysis, 170C and 370C induction, preparation for small
scale protein purification
DNA template
Cell disruption, fractionation, Ni-NTA
purification, SDS PAGE
Clones
Positive Clones
Expression Clones
Expression Cells
Purified Protein
Small ScaleExpression Screening
RESEARCH WORKFLOW AT -PPG
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LAB AND EQUIPMENTMolecular Biology Room (1094 SBC)
Three Thermocyclers. One RT-PCRIncubators: f loor and bench top
Centri fugesDNA electrophoresi s apparatus
Protein Prep & Pur. Room (1090)
Insect Cell Culture Room (1008)
Bio-interaction Analysis Room (1094)
AKTAPureAKTA Puri f ier
Many columnsSPEX Freezer Mi l l
SonicatorNano Drop
Centri fuges (50 µL-6000mL)Protein electrophoresi s apparatus
Bioneer Exiprogen Cel l Free System.
Biacore 3000 Surface Plasma Resonance system
Automated cel l counterCel l Cul ture Hood
Insect cel l shaker/Incubator
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Protein Expression Overview
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Full scale protein expression
Test several expression conditions
Your Gene
Affinity tagAssorted solubility partners available
Cleavage site for tag removal
PPG vector
Use PPG vectors or your own vector
https://psf.cobre.ku.edu/cores/ppg/about
http://www.infors-ht.com
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We use improved version of ligation independent cloning (LIC) for high throughput cloning. Any gene, any vector. No digestion, no ligation.
A gene can be cloned in a day.
Various fusion proteins are used to increase the expression level, the solubility and the stability of the protein or to drive the protein into inclusion bodies. All tags and fusion proteins are cleavable.
Met Affinity tag Fusion Protein Protease siteProtease site LIC Site & ORF
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PURIFICATION AND CHARACTERIZATION Affinity tags used: Histag >> GST tag >> others Columns used: Ni > SEC > IEX > hydrophobic Typical workflow:
Construct: HisTag—(other tag)—TEV site—Target Protein
Purification: Ni affinity (His-TEV cleavage) Ni SEC
Protein Characterization: SDS-PAGE, SEC, Native Gel (if requested)
Mass Spec (whole molecule, peptide digest)
Circular dichroism (prior to N-15 labeling for NMR)
Functional assays (usually by clients)
Western blot (usually by clients)
Thermo-shift assay
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Affinity Ion exchange
Hydrophobic interaction
Size exclusion Desalting
Imidazole gradient
Non-specific binding
Specific binding (your protein!)
Example Affinity Purification
Example Affinity Purification
Non-specific bindingCellular Proteins
Specific bindingYour Protein!!
AKTA PUREÄKTA PUREAutomated Purification System
http://www.gelifesciences.com
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A B C D E F G H I
SDS-PAGE gel of protein produce from the Cell-free system:A: pPG-EGFP; B: pTBSG-HDAC4.2; C: pTBSG-MEFA; D: pTBSG-hDBH; E: pREST; F: None; G: pBIEX-DBH; H: Bioneer GFP positive control. I: Protein Marker
Yield of soluble protein purified from 100 ul : A: 97ug; B: 80ug; C: 104ug; C: 124ug; D: 79ug; E: <5ug; F: <5ug; F: 100ug.
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Dextranmatrix
Gold layer
Glass
New Compound
OR
Dextranmatrix
Gold layerGlass
Modified from: Biacore Sensor Surface Handbook BR-1005-71 Edition AB
Biacore T200 Instrument Handbook 28-9768-63 Edition AC
https://psf.cobre.ku.edu/cores/ppg/about
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Other Services Provided Training of various techniques and equipment Western blotting and other expression assays Isotope labeling of proteins Proteins prepared for Mass Spectrometry,
NMR, X-ray crystallography Refolding and reconstitution of proteins Protein complex assembly Chemical modification of protein Protein interaction studies
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PHILIP GAO, PH. D.
DIRECTOR, COBRE PROTEIN PRODUCTION GROUP DEL SHANKEL STRUCTURAL BIOLOGY CENTER 2034 BECKER DRIVE, ROOM 1095 UNIVERSITY OF KANSAS LAWRENCE, KS 66047 PHONE: (785) 864-1825 (OFFICE)FAX: (785) 864-8141EMAIL: [email protected]
WEBSITE: HTTP://PSF.COBRE.KU.EDU/CORES/PPG/ABOUT
Contact Information
Projects related to PPG (publication, patent and Grant) can be arranged on collaborative basis.