Lab Director Fei Philip Gao, Ph.D. - hbc.ku.edu

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.

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.

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

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

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

Protein Expression Overview

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

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

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

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

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.

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

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

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.