SGC OxfordSGC Toronto SGC Stockholm

Experiences from SGC Stockholm

Helena BerglundStructural Genomics Consortium

Karolinska Institutet, Department of Medical Biochemistry and Biophysics Stockholm, Sweden

Structural Genomics Consortium

• not-for-profit organisation

• proteins with relevance to human health and disease

• three sites: Toronto, Oxford, Stockholm

• in Stockholm since spring 2005, ~25 people

closing down spring 2011

• Stockholm: > 2 deposited structures / month

• 180 pdb depositions, ~150 different structures

SGC-

Oxford

~65 staff

SGC-

Toronto

~70 staff

SGC-

Stockholm

~25 staff

board of directors

scientific committee

CEO

strategy:

• focus on protein production

• work flow

• biological background, optimisation and trouble shooting, follow up

•cloning, small scale expression screen, large scale culture and purif.

• bioinformatics, x-ray, biophysics expertise and infrastructure

•ELN electronic laboratory notebook (ConturELN)

•LIMS database - BeeHive

• selection of target families Stockholm• suggestions from us, Swedish scientific community, local Swedish scientific

committee

• approval from the SGC scientific committee

management and core scientist

biotechnology

structural biology teams

protein production SGC Stockholm:

•Stockholm main target areas: nucleotide metabolism, domains in apoptosis and receptor

signalling, PARPs, phosphoinositide and lipid signalling, amino acid metabolism, ATPases

• E. coli

• N-terminal His tag

• multi construct approach

• parallel processing of all constructs as far as possible

multi construct approach

• educated guesses of domain boundaries, vary from there

• different protein variants differ in

• expression

• purification yield

• crystallisation properties

• evaluated inGräslund et al. ”The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins” Protein Expr Purif. 2007 58, 210-221

Savitsky et al. ”High-throughput production of human proteins for crystallization : The SGC experience” J Struct

Biol. 2010 172, 3-13

targeted domain

Start Stop

AB

C

DE

evaluating the multi-construct approach:

a twice as many constructs could be

purified

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50

fraction of targets with at least one construct:

producing highly soluble protein

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4

8

12

16

20

resulting in well-diffracting crystals

all constructs

shortest &

full-length

all constructs

shortest &

full-length

a four-fold increase in well-diffracting crystals

all constructs (5008)

full-length and

shortest (925)

His tag:

MHHHHHHSSGVDLGTENLYFQ SM...

MGSSHHHHHHSSGLVPRG S....

• often tolerated in crystallization

• His tag removal - comparable with making new constructs

• SGC Stockholm :

~1/3 mounted crystals have the tag removed

-2008 -2011

deposited structures 77 143

tag removed 23 47

His tag making crystal contacts etc 3 ?

thrombin

TEV protease

His tag removal

• additional benefits

• increased purity after second IMAC to remove His tagged TEV protease

• allows more generous pooling from SEC

input cleaved target protein removed impurities & His tagged TEV

in flow trough protease rebound to resin

TEV-

protease

moving the His-tag to the C-terminus

• comparable with making new constructs

• between second half of 2008 and 2011 : 16 of ~80 new deposited structures

• initial but not good enough crystals with the N-His construct for most of them, tested new domain borders for a few

....AHHHHHH

non-cleavable C-term His6:

MHHHHHHSSGVDLGTENLYFQ SM....

from cleavable N-term His6:

purification

• IMAC-SEC over night on Äkta Xpress

• one set of buffers

20 mM HEPES, 300 mM NaCl, 10% glycerol, 1-2 mM TCEP

• final protein conc ≥15 mg/ml

gel filtration elution profiles as a quality indicator

Gräslund et al. Protein Expr Purif. 2007 Volume 58, Issue 2, April 2008, Pages 210-221

but over truncation!

gel filtration elution profiles

2

3

4,5

1

nice symmetric peak

continue as usual

single non-symmetric peak

continue as usual

desired protein in several separated peaks

continue with the lowest mol weight peak

(unless other knowledge about multimeric

state)

4: additional humps originate

from contaminating proteins

5: just broad and ugly

characterization

• SDS-PAGE

• contaminating proteins

• size heterogeneities

• Mass spectrometry

• verify the identity

• chemical homogeneity

• performed at Biotechnology KTH Stockholm

Harry Brumer

21.5

66.3

36.531.0

55.4

14.4

6.0

97.4

FTHFDB GMPR2 ITPAA

ITPKCA PAP39A

mass spectrometrydetected features:

• usually correct mass!

• point mutations

• construct/sample mix-ups

• His tag modification

• α-N-gluconoylation: + 178 Da

Geoghegan et al, Analytical Biochemistry 267 169-184 1999

178 Da

-28 Da

-2 x28 Da

-3 x28 Da

• co-purifying proteins

• E. coli: SlyD, GroEL, Fur, YfbG

• arg-lys substitutions

• differential usage of codons in humans

and E. coli

• E. coli might incorporate a lysine (AAA)

if there is a lack of correct arg-tRNA.

HO-CH2-[CHOH]4-CO-NH-R

mixed projects SGC Stockholm:

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60 70 80 90 100 110 120

purification scale-up problem

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60 70 80 90 100 110 120

I

M

A

C

S

E

C

ml

mAU

Audur Magnusdottir

model experiment

LYSATEBUFFER

His6-GFP pre-bound to

the top of the columns

scale-up problem

periplasm

removed

BUFFER LYSATE

• not due to :

reducing agent,

pH of lysate,

interferring DNA/RNA pieces,

HEPES buffer

competing E. coli proteins

.....

• work-arounds ?

• E coli lysate induced Ni-leakage

• metal chelators

scale-up problem

-30

-10

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40 60 80 100 120ml

mAU

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mAU

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mAU

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mAU

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mAU

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40 60 80 100 120ml

mAU

a b c

d e f

chaperone co-production

Baneyx & Mujacic, Nat Biotech (2004)

co-expression of the target proteins in E.

coli over expressing chaperones to obtain

higher amounts of soluble protein.

TaKaRa plasmid set

pG-KJE8 dnaK+dnaJ+grpE+groEL+groES

pGro7 groES+groEL s

pKJE7 dnaK+dnaJ+grpE x

pG-Tf2 groEL+groES+tig s

pTf16 tig

small scale expression screening

4 3 2 1purified proteins

scoring

0

50

100

150

200

250

-2 -1 0 1 2 3

nu

mb

er

of co

nstr

ucts

chaperone co-expression

difference small scale screening score

score(chaperone strain) - score(standard strain)

>380 constructs tested with different chaperones

48 constructs , 12 different targets side-by-side comparison using pG-

KJE8: dnaK+dnaJ+grpE+groEL+groES

5 structures until now

Martina Nilsson, Martin Hammarström

small set of constructs compared in large scale

positive effect generally lost

E. coli strain arctic express

Arctic Express (DE3) (Stratagene)

express Cpn60/Cpn10 from O.antarctica.

cold-adapted chaperones ( opt at 4-12 ºC )

1wf4.pdb

Oleispira antarctica

74% and 54 % aa identity to the E.coli GroEL/ES,

arctic express

difference small scale screening score

score(arctic express (DE3)) - score(standard strain)

42 constructs , 9 different targets, 4C

no deposited structure yet but 3.1 Å structure for one tricky

protein

PreScission protease TEV protease

GST-LEVLFQG HMHHHHHH-SGVDLGT-ENLYFQ SM.....

fusion proteins:

PreScission protease

Thioredoxin-LEVLFQG HMHHHHHH-SGVDLGT-ENLYFQ SM.....

TEV proteaseMBP-ETVRFQ S HHHHHH-SSGVDLGT-ENLYFQ SM....

TEV protease

constitutively produced, in vivo cleavage

TVMV protease

MHHHHHH-SSG-SHPQFEK-GT-ENLYFQ SM....

TEV protease

STREP-tag

difference small scale screening score

score(standard plasmid) - score(MBP)

still, 1 structure

Martin Hammarström

secreted proteins from E. coli:

The Novo Nordisk Foundation Center for Protein Research

– Newsletter 11

use E. coli for production of extracellular

di-sulfide bridge containing proteins

- benefits of periplasmic expression

- comparison between different three exporting

fusion proteins combined with two different

promoters

- araBAD promoter and OsmY

- described in Kotzsch et al : A secretory system for bacterial production of

high profile protein targets Protein Science 2011 20 597-609

Alexander Kotzsch, CPR

secreted proteins from E. coli

proteins in crystallisation

successful protein production

changing host:E. coli insect cells

produced, but

insoluble

all tricks tested

on protein family

members

Ida Johansson, Lionel Trésaugues

use of proteases :

limited proteolysis

TNKS2 hard to concentrate

added chymotrypsin during concentration

same construct borders but with C-terminal

His-tag concentrated well and diffracted to

2.5 Å

removed part of the N-terminal

opened B-loop loop -

best TNKS2 diffracting crystals ever : 1.95Å

Tobias Karlberg, Alex Flores

in situ proteolysis:

• addition of small amounts of protease ( 1: 200 - 1: 10000) in crystallisation

trypsin R or K, not before P

chymostrypsin F or Y or W , not before P

Glu-C V8 E or D

papain R or K

thermolysin L or F

endoproteinase Lys-C K = hydrophobic aa

Martin Welin

In situ proteolysis for protein crystallization and structure determinationNature Methods 2007 4: 1019 - 1021 work mainly from University of Toronto

In situ proteolysis to generate crystals for structure determination : an update PLoS one 2009 4:e5094

in situ proteolysis

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Targets Constructs Data sets Depositions

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6 4

chymotrypsin in crystallization

in situ proteolysis

SGC Stockholm

• tested on ~90 different targets

• usally one or two proteases

• usually not optimised protase ratio

• 5 structures

•trifunctional human enzyme encoded by the GART-gene

Linker (45) AIRS (475-792) Linker (15)GARS (1-430) GART 808-1010

solved by otherssolved at SGC

insoluble on its own

reductive methylation:

• di-methylation of all accessible lysine side

chains and the N-terminal amino group

• reduces surface disorder

Rayment Methods Enzymol. (1997). 276, 171

NH3+

-NH- -CO-CαH

CβH2

CγH2

CδH2

CεH2

NH

-NH- -CO-CαH

CβH2

CγH2

CδH2

CεH2

+CH3 CH3

reductive methylation

•expanded to 20-25 proteins, some crystals, lots of problems with precipitation

•no more structures

•large study Kim et al: Large-scale evaluation of protein reductuve methylation form improving

protein crystallization, 2008 Nature Methods 5 853-854

370 proteins tested - 7% success rate (structures solved)

Susanne van den Berg, Martin Högbom

DDX2A/eIF4A: 2G9N.pdb

DEAD domain of human DEAD-box helicase 2 DDX2A

• our first attempt using a kit from Jena Bioscience:

protein stabilisation:

• stabilisation by buffer exchange

• buffer composition, pH, salt,

additives - non specific protein

stabilizing agents, ligands

• ligand induced stabilisation

Flu

ore

scence Inte

nsity →

Temperature →

: SyproOrange fluoresce in low dielectric media (=non-polar environments

such as the interior of an unfolding protein or molten globule)

λex =490 λem =575nm

Pantoliano et al, Journal of Biomolecular Screening,2001;429-440

thermal shift assay: thermofluor

10 0

50 55 60 65 70 75 80 85 90

ΔT

thermal shift assays

• Performed in a RT-PCR instrument:

• 96 samples in ~1.5 hours, different buffers, ligands etc

• ~5-10 µg of protein/well, ~500 µg /96 well plate

• measure fluorescence as a function of temperature in each well

• fit each curve using a sigmoidal function

• extract Tm, transition temperature

• ΔTm -comparison between different conditions

••

knowledge based ligand screens

54 56 58 60 62 64

control

amino acids

Mg2+ATP,ADP,Mg2+ Mn2+

addition of ATP and Mn2+

enabled concentration

~20 combinations of amino acids,

ions, ATP, ADP•

•••

Tobias Karlberg

Human Glutamine Synthetase

2OJW

Lehtiö L et al (2009) J. Med. Chem. 52, 3108-3111

thermofluor curves of structure determined targetsGMPR2

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ITPKC

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TULP1

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FTHFDB

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IMPA2

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PRPSAP1

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PPP2R4

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NUDT3

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DDX25

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7000

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DDX2A

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PIP5K2C

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ITPA

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DRP2

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CPSF5

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FABP1

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alternatives

Guillermo Senisterra

Structural Genomics Consortium

University of Toronto

• only interpret good looking curves

• spin sample hard before thermofluor measurement

• alternative techniques:

Stargazer

for buffers: DLS

analytical SEC

buffer optimisation using DLS

• no crystals in standard buffer PARP13 catalytic domain at 1.1 Å 2x5y.pdb

Ann-Gerd Thorsell, Tobias Karlberg

buffer optimisation using DLS

DLS data at 0.2

mg/ml proteinstandard HEPES buffer pH 7.5 20 mM TRIS, 150 mM NaCl, pH 7.5

TNKS1Amonomers,

99% monodisp

monomers,

99.7% monodisp

PARP10Amonomers and dimers, some oligomers

and LA

monomers

98% monodisp

aggregation with time (1-2 h)

PARP12Aoligomers

98.5% monodisp

multimers

99.4% monodisp

PARP15A dimers and monomers dimer or monomer 97.5% monodisp

PARP13A dimers, tetramers and aggregates >98% monomers,

Natalia Markova

What will happen now?

• Toronto Oxford still running

• for all deposited:

pdb

M&M

reagents

• for many of them:

iSee data packs

• for some additional proteins:

protein production protocols

• publications

• visiting scientist program

what will happen now?

Stockholm site

biotechnology structural biology teamsx-ray

ACKNOWLEDGEMENTS

SGC Stockholm :Aida SehicAlex FloresAlexander KotzschAndreas JohanssonAnnette RoosAnn-Gerd ThorsellAudur MagnusdottirBiljana JovanovicBjörn ForsströmBrendan McManusCamilla PerssonDerek OggElisabeth WahlbergGlareh AskareihHanna WillanderHelena BerglundHerwig Schüler

Ida JohanssonJessica AnderssonJohan NilvebrandtJohan WeigeltJohanna SagemarkJonas UppenbergKarin WalldénKate KouznetsovaKenneth OlesenKerstin MichalkeLari LehtiöLasse DahlgrenLinda SvenssonLionel Trésaugues Lola HermanLouise EgebladLovisa Holmberg-SchiavoneMagdalena Wisniewska

Mailen AnderssonMalin UppstenMarina SiponenMartin HammarströmMartin HällbergMartin HögbomMartin MocheMartin Welin Martina NilssonMonika AbramczukNatalia MarkovaNatalie PerssonPatricia WennerstrandPatrick SchützPer KraulisPetra Nilsson-EhlePetri KursulaPetter NorbergPål Stenmark

Pär NordlundRobert BusamRuairi CollinsStephan KolStina LundgrenSusanne FlodinSusanne GräslundSusanne van den BergTanya KotenyovaTine Kragh-Nielsen Tobias KarlbergTomas Nyman Torun EkbladUlrika ErikssonUlrika IsbergUrzula KosinskaÅsa Kallas

SGC TorontoSGC Oxford

FUNDING PARTNERS

Canadian Institutes for Health Research, Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Knut and Alice Wallenberg Foundation, Merck & Co., Inc., Novartis Research Foundation, Ontario Innovation Trust, Ontario Ministry for Research and Innovation, Swedish Agency for Innovation Systems, Swedish Foundation for Strategic Research, and Wellcome Trust. www.thesgc.org