pCOLD Vectors and Other

Alternative

Expression Systems

for Structural Genomics

Optimal Growth Acclimation Steady Low Temp Growth Stationary Phase Adapted Cells Phase

Non-CSPs

Non-CSPs

CSPs

CSPs

Growth Curve

Cold- shock adaptation of E. coli

< 20 °C37ºC

pCold I (4.4 kb)

lacI

ColE1 ori

Am

p

M13 IG

cspA promoterlac operatorcspA 5’UTRTEEHis6

multiple cloning sitecspA 3’UTR

factor Xa site

TEE (translation enhancing element): ATGAATCACAAAGTG (MNHKV)His6: CATCATCATCATCATCATFactor Xa site: ATCGAAGGTAGG (IEGR)Multiple cloning site:

CATATGGAGCTCGGTACCCTCGAGGGATCCGAATTCAAGCTTGTCGACCTGCAGTCTAGA

NdeI SacI KpnI XhoI BamHI EcoRI HindIII SalI PstI XbaI

Maps of pCold vectors

pCold III (4.4 kb) la

cI

ColE1 ori

Am

p

M13 IGlac operatorcspA 5’UTRTEE

multiple cloning sitecspA 3’UTR

cspA promoter

pCold IV (4.4 kb) la

cI

ColE1 ori

Am

p

M13 IGlac operator

cspA 5’UTRmultiple cloning sitecspA 3’UTR

cspA promoter

pCold II (4.4 kb)

M13 IG

Am

p

ColE1 ori

lacI

lac operatorcspA 5’UTRTEE

His6

multiple cloning sitecspA 3’UTR

cspA promoter

T S1 2

trigger factor

EnvZ-B

T S1 2

T S

calmodulin

1 2

1 2 3 4 5 0 12 24 36 48 hr

EnvZ-B

1 2 3 4 5 6 7

SDS-PAGE of whole cell lysates - E.coli EnvZ ATP-binding domain (EnvZ-B), Xenopus calmodulin (CaM) and E.coli trigger factor expressed using pCold vectors

EnvZ-B

Expression level and solubility of different proteins - pColdI and pET14 systems

In total, 38 genes as shown above were chosen and cloned in pColdI and pET14 vectors, respectively. Samples with better expression level and/or solubility in pET14 were labeled with blue color, and red color for those with better expression and/or solubility in pColdI. NS: not soluble; NE: no expression; NA: not available.

Human genes

Drosophila genes

pET14 pColdIgene

expression solubility expression solubility

FR2FR4FR5FR6FR14FR37FR48FR59FR70

++NE++

+++

NE+++++

+NA+

NS

++NA

+++

++

+++NE

++ NS

++ +++++

+++++++

++ +++ ++

++

NA++

FR78

NE NA NE NA

+++ ++ +++ ++

C.elegans genes

E.coli genes

pET14 pColdIgene

expression solubility expression solubility

ER6ER7ER15ER19ER64ER85ER115ER130ER135

++++

++++NE+

+++++++

NSNSNS+++NA

NS+++

+

+++++

+++NE NA

NS++++

++NS+

+ ++ +++

NS+

NS+++

+++

pColdIpET14gene

expression solubility expression solubility

HR8HR31HR520HR521HR522HR524HR529HR535HR540

+++

NE++

+++

+++NSNANS

NA NE

++

NE++

NS

NA

NSNA

+++NA

++++++NA

++ NS NA

NE NA NS

NE

NS

+++

NENE

++

NE

pET14 pColdIgene

expression solubility expression solubility

WR13WR24WR26WR27WR33WR35WR41WR44WR49

+++NE+++++++++++++++NE+++

NSNANSNS+++

NS++

+++

+++

++NE

++++++ +++

NS+++

+++NSNA

NS +++ NS

NS

NANSNS

WR53 +++ +++ +++ +++

Purified protein NMRCell lysate NMR

[1H-15N]-HSQC spectra of 15N-enriched Xenopus calmodulin produced with pCold vector

Sequential connectivity map summarizing the results of triple-

resonance NMR experiments with calmodulin in whole cell lysates

~ 80% of the peaks are assigned

Target Selection, Cloning and Protein Production

Validate/ cDNA

Identify Target ORF

Clone into Expression Vectors

Human, C. elegans, Drosophila, Arabidopsis, yeast and others

N/C-Terminal His-Tags, pCold, No tag, MBP, SUMO, Pichia, cell-free

Small Scale Expression

Large Scale Fermentation/Protein Purification

Soluble

Insoluble ornot expressed

Multiplex Expression System

Classical Restriction Endonuclease/Ligase-

dependent cloning

E. coli Expression Vectors

Eukaryotic Expression systems

Expanded multiplex system

1) Gateway MBP-fusion expression system (Kapust & Waugh 1999) (collaboration with D. Waugh, NCI)

2) SUMO system (Lifesensors, Inc. Malvern, PA) (collaboration with T. Butt, Lifesensors, Inc.)

Attempt to use fusion protein expression systems in place of our standard T7 Multiplex Expression System (Acton et al, submitted) for a setof 50 eukaryotic target proteins

MBP fusion coupled with cleavage by TEV

MBP is cleaved from its fusion partnerby Tobacco Etch Virus (TEV) Proteinase

TEV recognizes the consensus sequence:

Glu-X-X-Tyr-X-Gln-Ser

(Routzahn & Waugh 2002)

Both in vivo and in vitro cleavage conditionsare being investigated

Interesting note: Recombinant TEV does not fold properlyin vivo and it must be generated as an MBP fusion as well

(Dougherty et al., 1989)

Summary of MBP screening

N/ANENENEWR15

Y E/SE/SE/SWR14

 N/AE/SE/NSE/NSWR13

Y E/SE/SE/SWR11

 lowE/SNENEWR10

N/ANENENEWR9

Y E/SNEE/SWR8

Y E/SNENEWR6

N/ANENENEWR5

Y E/SE/SE/SWR4

YE/SE/SE/NSWR3

 lowE/SNEE/NSWR2

MBP-fusion in vitro cleavage

MBP-fusion

uncleaved

MBP-fusion in vivo

cleavage

pETTarget

Not expressed

Expressed/soluble

Expressed/insoluble

N/ANENEE/NSWR54

Y E/SE/SE/SWR53

 lowE/SE/SE/NSWR49

N/ANENENEWR44

 N/AE/SE/SE/SWR43

 YE/SE/SE/SWR41

 lowE/SE/SE/SWR39

N/ANENENEWR28

 lowE/SE/NSE/NSWR27

 N/AE/SNEE/NSWR26

 lowE/SPEPEWR18

N/ANENENEWR16

MBP-fusion in vitro cleavage

MBP-fusion

uncleaved

MBP-fusion in vivo

cleavage

pETTarget

SUMO system

• SUMO is a Ubiquitin-like (UBL) protein• UBLs are small, highly soluble, globular proteins• SUMO has been reported to exhibit chaperone-like activity• Ulp1 is used to cleave the protein target from the fusion by recognizing the entire SUMO protein• SUMO system utilizes Ni-affinity chromatography• Cleavage must occur in vitro

(Figure courtesy of www.lifesensors.com)

6хHis SUMO Protein of interest

SUMP protease (Ulp1)

Summary of SUMO fusion screening

Target IDpET

(N-terminal His-tag)SUMO fusion

AR5 NE NE

AR22 E/S E/S

FR10 E/NS E/S

HR894 E/NS E/NS

HR1553 E/NS E/S

HR1686 E/NS E/NS

HR1697 NE E/S

WR26 E/NS E/NS

Not expressed

Expressed/soluble

Expressed/insoluble

A stable cell-free protein synthesis system prepared from wheat germ

Improvement in preparing cell extracts: removing endosperm contaminants, including tritin, thionin, ribonucleases, deoxy ribonucleases, and proteases, by thorough washing and sonication. (Proc. Natl. Acad. Sci. USA 99, 14652-57)

Protein synthesis in the dialysis system. (A and B) Coomassie blue-stained SDS polyacrylamide gels showing DHFR synthesis with (A) or without (B) addition of new mRNA. Arrows and asterisks mark DHFR and creatine kinase, respectively. (C) Amounts of DHFR synthesized as determined from densitometric scans of the gels in A (closed circles) and B (open circles).

DHFR

CK

(collaboration with Y. Endo, Ehime University)

A cell-free expression vector and its performance

GFP

mRNA supplement (92 g in 500 l reaction each time)

(a) Schematic illustration of pEU. (b) SDS/PAGE analysis of GFP produced during 14 days of reaction. mRNA produced by transcription of circular pEU was used for the translation reaction in the dialysis membrane system and was added every 48 h. A 0.1-µl aliquot of the mixture was run on the gel, and protein bands were stained with CBB. The arrow shows GFP; "st" designates an authentic GFP band (0.5 µg).

****

*

*

*

* *

Protein synthesis of human targets by wheat-germ system

Target ID

pET(N - terminal tag) Protein synthesis by wheat -

germ system

Expression Solubility Expression Solubility

AR15 +++ + ++ ++ AR16 NE NA ++ ++

AR21 NE NA + +++ AR22 + +++ +++ 0.5 FR10 +++ NS + +++ HR812 NE NA +++ ++ HR894 +++ NS NE NA HR919 +++ NS ++ +++ HR945 +++ NS NE NA HR969 +++ ++ ++ +++ HR1553 +++ NS NE NA HR1576 0.5 NS NE NA HR1686 +++ NS ++ ++ HR1697 NE NA NE NA HR1719 +++ NS + +++ HR1722 +++ NS + + HR1738 +++ NS NE NA WR13 +++ NS +++ 0.5 WR19 +++ NS +++ 0.5 WR20 NE NA +++ NS WR21 +++ + 0.5 NS WR23 +++ NS +++ NS WR24 NE NA NE NA WR26 +++ NS +++ NS WR27 +++ NS +++ 0.5 WR35 +++ NS +++ +++ WR38 ++ NS +++ 0.5 WR41 +++ +++ +++ +++ WR43 + ++ + + WR44 NE NA +++ NS WR50 + NS ++ ++ WR53 +++ +++ ++ +++ WR54 +++ NS ++ +++

Summary of screening by cell-free system from wheat germ

Not expressed

Expressed/soluble

Expressed/insoluble

1 AR5 NA NA NA NA2 AR153 AR16 NA NA NA4 AR21 NA5 AR22 NA6 FR4 NA NA7 FR108 FR599 HR79 NA10 HR9111 HR547 NA NA12 HR812 NA NA NA13 HR894 NA NA14 HR91915 HR945 NA NA16 HR96917 HR1553 NA18 HR1576 NA NA19 HR1686 NA20 HR1697 NA NA NA21 HR171922 HR1722 NA23 HR1738 NA NA24 HR185425 HR1869 NA26 HR1889 NA27 HR191328 HR1953 NA NA29 WR12 NA30 WR1331 WR1932 WR20 NA33 WR2134 WR2335 WR24 NA NA NA NA36 WR26 NA NA NA37 WR2738 WR28 NA NA39 WR35 NA40 WR3841 WR40 NA NA NA42 WR4143 WR42 NA NA NA44 WR4345 WR44 NA NA NA NA46 WR4947 WR50 NA48 WR51 NA49 WR5350 WR54 NA NA

LEGEND:

= not attempted yet = hook up complete H = hooked up = expressed E = expression tested = not expressed S = solubility = insoluble = soluble

NA = not applicable = in progress = expressed & soluble in at least one system

Wheat germ extract

H E SSS

E. coli pET (Cter-H 6)

H E

E. coli pCOLD vector

H E SNumber Target ID H

Eukaryotic Core 50

E S

E. coli pET (Nter-H 6) E. coli MBP (uncut)

H E S

E. coli MBP + TEV

H E

E. coli SUMO fusions

H E S

P. pastoris pPIC3.5

H E S

Panel of 50 eukaryotic targets in eight expression systems

Acknowledgements

Guoliang Qing Thomas Acton Tatsuya SawasakiLi-Chung Ma Rong Xiao Yaeta Endo Ahmad Khorchid Ritu Shastry Kate Drahos G. V. T. Swapna Chi Kent Ho Tauseef R. ButtTapas K. Mal Natalia Denissova David S. WaughMasanori Mitta Takayama Bonnie CooperBing Xia Kellie CunninghamSangita Phadtare Liang-yu (Lydia) ShihHaiping Ke Yi-Wen Chiang Gaetano T. Montelione Shin-Geon ChoiMitsuhiko Ikura Masayori Inouye

What if…?

PCR & cloning of ORFs Construct validation

Small-scale expression & solubility screens

Unexpressed or Insoluble

Proteins

Protein Production Pipeline

PCR & cloning of ORFs Construct validation

Small-scale expression & solubility screens

using T7 system

Analysis & passing oftargets to fermentation

Large-scale production &purification of 15N/13C, or selenomethionine-labeled proteins

Structure determination by NMR

Structure determinationby X-ray crystallography