Optimizing Efficiency and Effectiveness when Optimizing Efficiency and Effectiveness when Expressing Proteins for Drug Discovery Expressing Proteins for Drug Discovery

Krista BowmanGenentech, Inc.

May 3, 2012

OutlineOutline

• Structural Biology Protein Expression Group– Our goals and general approaches to expression

• Analyzing Protein Expression– Automated affinity Phytip capture/elution– Fluorescent Size Exclusion Chromatography (fSEC)

1

– Fluorescent Size Exclusion Chromatography (fSEC)

• Tricks to deliver much needed proteins– Deglycosylated proteins for structural analysis– Co-expression of kinases with phosphatases– Baculovirus particles for displaying membrane

proteins

SB Protein Expression Group: MissionSB Protein Expression Group: Mission

•• Facilitate Discovery of Novel TherapeuticsFacilitate Discovery of Novel Therapeutics

•• Understand Protein Structure, Function, and RegulationUnderstand Protein Structure, Function, and Regulation

DiscoveryResearch reagents

Small MoleculeDiscovery

22

Structural BiologyExpression Group

Antigens

Research reagentsDiscovery

StructuralBiology

Our ObjectivesOur Objectives

• Provide high quality protein expression systems– Baculovirus and E. coli systems for research reagents– BacMam for cell-based assays – Protein Structures

• Research collaborations

33

• Research collaborations– Individual research investigators

• Process innovations and new technologies– Efficiency and effectiveness – Earlier data-driven decision-making– Expand capabilities and functions

Process Innovations for Greater ImpactProcess Innovations for Greater Impact

6-12Constructs

EXP PUR Crystal trialsAnalyze

Classical approach

EXP48-96

PURAnalyze Crystal trials

Higher impact approach

44

• More shots on goal• Earlier protein characterization and triage• Fewer large scale purification/characterization

EXP48-96

Constructs PURAnalyze Crystal trials

Automated HighAutomated High--Throughput SubcloningThroughput Subcloning

N-tag C-tag No Tag

> 74 Expression Vectors

Restriction-independentcloning system

Multiple constructs per target

55

HIS FLAG GST

Automated DNA purification(Beckman FX)

“Lysate Direct Phytip Purification”co-development with Phynexus

(manuscript in preparation)

Yvonne Franke

E. coli, BEVS, pRK, BacMam

Preparing Preparing BaculoviralBaculoviral StocksStocks

Co-transfectionsAmplifications

Expression Analysis

6

Adherent (800 uL) Suspension (4 mL) Expression stocks

Virus Titer

Using Flow Cytometry to Characterize InfectionUsing Flow Cytometry to Characterize Infection

Infected

UninfectedControl

24hrs Post

Infection

Gp64 - fluorescence

G

P

6

4

PE

488 585

Cell

Co

un

t

Infected cells

77

Gp64 - fluorescenceInfected cell

Gp64-PE-Fluoresence

Infection (0hr) Harvest

Via

bil

ity (

7-A

AD

)

E. Coli expression Host strains Induction method Promoters, tags Domain boundaries

E. Coli expression Host strains Induction method Promoters, tags Domain boundaries

Construct Triage by Construct Triage by QuantitativeQuantitative Expression AnalysisExpression Analysis

Expression Analysis

Explore/optimize multiple expression variables simultaneously

5 mL culturesDomain boundaries Domain boundaries

88

Phynexus tips

Baculovirus Expression Sf9 and T ni cellsMOI, harvest time Promoters, tagsDomain boundaries

5 mL cultures

Detergent Screening for Detergent Screening for SolubilizationSolubilization (5 mL cultures)(5 mL cultures)

None DDM Triton Brij LDAO FC12

Lysis(detergent) % detergent

S200

monomer?

aggregate

9

None DDM Triton Brij LDAO FC12

48 automated purifications to screen lysis conditions – 1 hr

Construct Triage: Extracellular proteins (5 mL cultures)Construct Triage: Extracellular proteins (5 mL cultures)

1 2 3 4 1 2 3 4 Gravity flow Phynexus

Ni-NTA

1 2 3 4 1 1 2 2 3 3 4 4

Automated method for secreted proteins with 3 different tags

10

1 mL Phytip5-10 uL resin

Phynexus MEAMicro-Extractor

Automated Instrument

48 automated purifications from 5 mL culture each in 8-10 hrs

FLAG

Fc(MAbSuRe)

1 2 3 4 1 1 2 2 3 3 4 4

1 2 1 1 2 2

Can We Predict Crystallization Success Earlier?Can We Predict Crystallization Success Earlier?

Best predictors using purified protein:Aggregation state – non-aggregatedDispersity – homogeneous, non-proteolyzed

Size ExclusionChromatography Light Scattering

1111

Q: Can we rapidly measure aggregation state,homogeneity, and yield in cell lysates?

Chromatography

Fluorescence-basedSize Exclusion Chromatography

Construct Triage by Fluorescence SECConstruct Triage by Fluorescence SEC

NTA

Binds 6xHis Tag

Detection

Cells Expressing Target Protein

Cells Expressing Target Protein Lyse, clarify

Add label

1212

FluoresceinNTA

NTA

Scaffold: ala Szoka et al,

Bioconjugate Chemistry, 2006

6xHis

Fluorescent

Separate by sizemonitor fluorescence

Protein X “Visible” in Cell Lysate by FSECProtein X “Visible” in Cell Lysate by FSEC

M L

A280 A

bso

rban

ce

Size Exclusion Chromatography

M L FT WASH E1E2

FSEC, Cell Lysate

•Monodisperse•Monomeric•High-yield

FSEC, Cell Lysate

•Monodisperse•Monomeric•High-yield

Flu

ore

scen

ce

Identical Traces:•Lysate

•Purified

Flu

ore

scen

ce

13

IMAC (6xhis) Purification

A280 A

bso

rban

ce

A280 SEC of Lysate: •Uninformative

A280 SEC of Lysate: •Uninformative

Flu

ore

scen

ce

Flu

ore

scen

ce

Early Decision: deEarly Decision: de--prioritize Constructprioritize Construct6xHis-GST vs 6xHis: Size exclusion chromatography

Fluorescence

1414

WellWell--Behaved ProteinsBehaved Proteins

DC

B

B AAmono-disperse

free probe

aggregate

15

D

C

Less WellLess Well--Behaved ProteinsBehaved Proteins

F

G

H

I

A B C D FE

aggregate

16

A

B

C

D

E

H IG

monodisperse

free probe

6xHis

Fluorescent

Expression levelAggregation stateBehavior on SECProtein complexes

Measuring Protein Quality and Quantity from 5 mL culturesMeasuring Protein Quality and Quantity from 5 mL cultures

protein quantity protein quality

17

• Routine use in expression analysis

• Earlier data-driven decisionsB

A

A Baggregate

probe

fSEC identifies high, aggregated protein expression

NN--linked Glycosylationlinked Glycosylation--deficient Protein for Structuresdeficient Protein for Structures

Complex or hybridglycans

N-acetylneuraminic acid

Galactose

Glucose

Mannose

N-acetylglucosamine

Fucose

Kifunensine(IC50 20 nM)

Endo H-

sensitive

Endo H-

insensitive

Swainsonine(IC50 100 nM)

1818

Mannosidase IGlnT1

(N-acetylglucosaminyl-transferase)

glycans

“paucimannose”

Mannosidase II

N-acetylglycosaminidase

ER golgi

Mannosidase1A, 1B

XX X

Protein Weight Loss PlanProtein Weight Loss Plan

kif - + + + endo H - - + ++

38

49

62

98

Endo H

Target protein production

• 2 mg/mL kifunensine

1919

1 GlcNAc (MS data)

17

14

28

38 Endo H• 2 mg/mL kifunensine

• Co-expression with Endoglycosidase H

V. Change et al. Structure (2007) 15:267

C. Garcia (personal communication)

“more homogeneous” protein

Expression of Expression of deglycosylateddeglycosylated SeSe--metmet--labeled Proteinlabeled Protein

Kif/EndoH

Glycosylated Kif/EndoH

M T W Th F

Mediaexchange 1:1 infect

100 ug/mLSe-Met

100 ug/mLSe-Met harvest

82% Se-Metincorporation

20

Kif/EndoH+ -

S75 10/30, TBS

diffraction to < 2.5A

Patrick Lupardus

ATP: - + - + - +

kd ubl sdd

pSer

kinase domain(1.9Å resolution)

Kinase crystal structure and biochemistryKinase crystal structure and biochemistryCo-expression with

phosphatase PPP1CASe-Met labeling

(70-80% incorporation)

21

Active kinase panel enabled dissection of domain contributions:

• auto-activation• substrate specificity• enzyme kinetics

WB: Anti-pSerXXX

kd ubl

kd

Xiaolei Ma, Elizabeth Helgason, Melissa Starovasnik, Erin Dueber

Cy5-label

Protein X on Microarray

Y-displaying-baculovirus

Baculovirus Particles for Protein DisplayBaculovirus Particles for Protein DisplayProof of Concept: CD200 -- CD200R

0

100

200

300

400

500

Average SAR

Bait: CD200-Protein A beads

CD200RHit 3 times

22Lino Gonzalez, Irene Tom

SPDI Microarray

• Similar hits: Baculovirus andprotein A bead display methods

• Other protein interactions confirmed

• Screen protein microarray withmembrane-bound proteins

0

500

1000

1500

2000

2500

3000

Average SAR

Bait: CD200-virus

CD200RHit 3 times

AcknowledgementsAcknowledgementsSB Protein Expression Bobby BrillantesMary CoonsTrisha Dela VegaAlberto EstevezYvonne FrankeJiyoung HwangRekha IyerKyle Mortara

Structural BiologyGladys De LeonPaola Di LelloCharlie EigenbrotRina FongSeth HarrisSarah HymowitzJim KieferChris Koth

Susmith MukundJeremy MurrayAngela OhBorlan PanLionel RougeMelissa StarovasnikMark UltschHeidi WallweberWeiru Wang

2323

Kyle MortaraJian PayandehAlex RondonJun SampangStephanie ShriverChristine TamInna Zilberleyb

Chris KothPatrick LupardusTill MaurerRobert Mayer

Oligo Synthesis Research CollaboratorsDNA Sequencing Small Molecule ProgramBioinformatics Project teamsProtein Chemistry

Weiru WangPeter YinChristine YuPing Wu