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

    Krista Bowman Genentech, 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

    Discovery Research reagents

    Small Molecule Discovery

    22

    Structural Biology Expression Group

    Antigens

    Research reagentsDiscovery

    Structural Biology

  • 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

    6-12 Constructs

    EXP PUR Crystal trialsAnalyze

    Classical approach

    EXP 48-96

    PURAnalyze Crystal trials

    Higher impact approach

    44

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

    EXP 48-96

    Constructs PURAnalyze Crystal trials

  • Automated HighAutomated High--Throughput SubcloningThroughput Subcloning

    N-tag C-tag No Tag

    > 74 Expression Vectors

    Restriction-independent cloning 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-transfections Amplifications

    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

    Uninfected Control

    24hrs Post

    Infection

    Gp64 - fluorescence

    G

    P

    6

    4

    PE

    488 585

    C e ll

    C o

    u n

    t

    Infected cells

    77

    Gp64 - fluorescence Infected cell

    Gp64-PE-Fluoresence

    Infection (0hr) Harvest

    V ia

    b il

    it y (

    7 -A

    A D

    )

  • 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 cultures Domain boundaries Domain boundaries

    88

    Phynexus tips

    Baculovirus Expression Sf9 and T ni cells MOI, harvest time Promoters, tags Domain 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 Phytip 5-10 uL resin

    Phynexus MEA Micro-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-aggregated Dispersity – homogeneous, non-proteolyzed

    Size Exclusion Chromatography Light Scattering

    1111

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

    Chromatography

    Fluorescence-based Size 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

    Fluorescein NTA

    NTA

    Scaffold: ala Szoka et al, Bioconjugate Chemistry, 2006

    6xHis

    Fluorescent

    Separate by size monitor fluorescence

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

    M L

    A 2 8 0 A

    b s o

    rb a n

    c e

    Size Exclusion Chromatography

    M L FT WASH E1E2

    FSEC, Cell Lysate

    •Monodisperse •Monomeric •High-yield

    FSEC, Cell Lysate

    •Monodisperse •Monomeric •High-yield

    F lu

    o re

    s c e n

    c e

    Identical Traces: •Lysate

    •Purified

    F lu

    o re

    s c e n

    c e

    13

    IMAC (6xhis) Purification

    A 2 8 0 A

    b s o

    rb a n

    c e

    A280 SEC of Lysate: •Uninformative

    A280 SEC of Lysate: •Uninformative

    F lu

    o re

    s c e n

    c e

    F lu

    o re

    s c e n

    c e

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

    Fluorescence

    1414

  • WellWell--Behaved ProteinsBehaved Proteins

    DC

    B

    B AA mono-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 level Aggregation state Behavior on SEC Protein 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 B aggregate

    probe

    fSEC identifies high, aggregated protein expression

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

    Complex or hybrid glycans

    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 I GlnT1

    (N-acetylglucosaminyl- transferase)

    glycans

    “paucimannose”

    Mannosidase II

    N-acetylglycosaminidase

    ER golgi

    Mannosidase 1A, 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

    Media exchange 1:1 infect

    100 ug/mL Se-Met

    100 ug/mL Se-Met harvest

    82% Se-Met incorporation

    20

    Kif/EndoH + -

    S75 10/30, TBS

    diffraction to