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A PROTEOMICS BASED APPROACH TO THE REGULATIONA PROTEOMICS BASED APPROACH TO THE REGULATION

THE SMALL HEAT SHOCK PROTEINS OF DROSOPHILATHE SMALL HEAT SHOCK PROTEINS OF DROSOPHILAJudite Dias1, Edgar F. da Cruz e Silva1, Pedro Domingues2 and Odete A.B. da Cruz e Silva1

1Centro de Biologia Celular and 2Departamento de Qumica, Universidade de Aveiro, 3810-193 Aveiro, Portugal

Judite Dias1, Edgar F. da Cruz e Silva1, Pedro Domingues2 and Odete A.B. da Cruz e Silva1

1Centro de Biologia Celular and 2Departamento de Qumica, Universidade de Aveiro, 3810-193 Aveiro, Portugal

The expression levels of small heat shock proteins are known to fluctuate in response to various stressors including high temperatures and toxic compounds. They may also play distinct functional r

normal development. Four small heat shock proteins have been identified in D. melanogaster(hsp22, hsp23, hsp26 and hsp27), that share high sequence homology. However, their expression patterns d

the organs and developmental stages, each showing specificity of expression and intracellular localization. Hsp27 is expressed throughout the whole life cycle, while hsp22 expression is known to change

the absence of stress. The main objective of this work is to express polyhistidine-tagged Drosophila hsp27 and hsp22 in bacterial systems, followed by subsequent Ni chromatography affinity purification.

Drosophila binding proteins by a proteomics based approach, including 2D gel electrophoresis and mass spectrometry, fly lysates will be passed through hsp columns and the bound proteins eluted and c

identification of novel protein-protein interactions should provide interesting insights into the physiological role and regulation of the Drosophila small heat shock proteins.

HspHsp

expressionexpression

HspHsp

expressionexpression PurificationPurificationPurificationPurification

Protein-Protein-

proteinprotein

interactionsinteractions

Protein-Protein-

proteinprotein

interactionsinteractions

MassMass

SpectrometrySpectrometry

MassMass

SpectrometrySpectrometry

Imunoblotting of Drosophila

lysates

A sample of flies was incubated at

28 C for 20 minutes and

subsequently at 37 C for 100

minutes. Half of this sample was

given a recovery time of about 16

hours. These samples were frozen

at 80 C and homogenised in 1%

SDS. They were loaded into an

SDS- acrylamide gel and

transferred into a nitrocellulose

membrane. The blots were

incubated with -HSP22 and -

HSP27 antibodies and developed

with NBT/BCIP.

Transformation

The cDNA form HSP22 was

cloned into a pET30 vector

(Novagen) containing a

polyhstidine tag. The same

procedure was used with HSP27.

Each vector was transformed into

two distinct strains: GJ1158 and

BL21DE3.

Protein expression

The transformed GJ1158 was

grown in LBON until the OD (600

nm) had reached 0.6. At this point

NaCl was added in order to induce

the protein expression. The

transformed BL21DE3 was grown

in LB and the protein expression

was induced with IPTG. These

bacterial cultures were centrifuged

and the pellets were frozen at 80

C.

Protein expression

The transformed GJ1158 was

grown in LBON until the OD (600

nm) had reached 0.6. At this

point NaCl was added in order to

induce the protein expression.

The transformed BL21DE3 was

grown in LB and the protein

expression was induced with

IPTG. These bacterial cultures

were centrifuged and the pellets

were frozen at 80 C.

Purification

Cell pellets were ressuspended

in Binding Buffer, to which PMSF

had been added, and sonicated.

The NI-NTA column (qiagen)

was equilibrated and the cell

lysate was loaded into it.

Afterwords the column was

washed with Binding Buffer and

Washing Buffer. The protein was

then eluted with Elution Buffer.

All fractions were collected in

order to perform an SDS-PAGE.

Affinity Chromatography

The purified HSP is dialised

with Binding Buffer and religated

to the NI-NTA column. A

Drosophila homogenate is then

passed through the column and

this protein set is eluted with

elution buffer.

transformation

cell

lysate

Ni-NTA

column

Purified

HSP

Religation

Affinity

Chromatography

2D-SDS-PAGE

Mass

Spectrometry

ResultsResultsResultsResults

Induction of endogenous HSP productionInduction of endogenous HSP production

Probing with -hsp27 antibody Probing with -hsp22 antibody

hsp27hsp27

hsp22hsp22

M + + - + M + - + + H e a t S ho c k

M - + - - M - - + - R e c o v e ry T im

Protein purificationProtein purification

BL21 DE3-pet-hsp22 GJ1158-pet-hsp

celllysate

Flowthrough

Washwith

BB

Washwith

WB

Elution

Strip

Dialysis

M

celllysate

Flow

through

Washwith

BB

Washwith

WB

Affinity chromatographyAffinity chromatography

ConclusionsConclusions

So far the expression and purifica

tagged HSP22 and HSP27 have been do

The next goal is to optimize the condition

chromatography in order to procced with t

of the proteins that interact with these HSP

Joanisse, D.; Inaguma, Y.; Tanguay, R. (1998) Clonin

expression of a nuclear ubiquitin conjugating enzyme (D

with small heat shock proteins in Drosophila melanogast

Res Commun, 244:102-109.

Joanisse, D.; Michaud, S.; Inaguma, Y.; Tanguay, R. (19 proteins of Drosophila: Developmental expression and

23:369-376.

Marin, R.; Tanguay, R. (1996) Stage-specific localizat

shock protein Hsp27 during oogenesis in Drosophila m

105:142-149.

Mehlen, P.; Briolay, J.; Smith, L.; Diaz-Latoud, C; Fabre

A.P. (1993) Analysis of the resistance to heat and hydroge

COS cells transiently expressing wild type or delet

Drosophila 27 Kda heat shock protein. Eur. J. Biochem, 21

Morrow, G.; Inaguma, Y.; Kato, K.; Tanguay, R. (2000) T

protein Hsp22 of Drosophila melanogaster is a M

Displaying Oligomeric Organization. J Biol Chem, 275:31

Tanguay, R.; Joanisse, D.; Inaguma, Y.; Michaud, S. (19

proteins: in search fro functions in vivo. In: Storey KB

Stress and Gene Regulation. BIOS Scientific Publishers

138.

Joanisse, D.; Inaguma, Y.; Tanguay, R. (1998) Clonin

expression of a nuclear ubiquitin conjugating enzyme (D

with small heat shock proteins in Drosophila melanogast

Res Commun, 244:102-109.

Joanisse, D.; Michaud, S.; Inaguma, Y.; Tanguay, R. (19 proteins of Drosophila: Developmental expression and

23:369-376.

Marin, R.; Tanguay, R. (1996) Stage-specific localizat

shock protein Hsp27 during oogenesis in Drosophila m

105:142-149.

Mehlen, P.; Briolay, J.; Smith, L.; Diaz-Latoud, C; Fabre

A.P. (1993) Analysis of the resistance to heat and hydroge

COS cells transiently expressing wild type or delet

Drosophila 27 Kda heat shock protein. Eur. J. Biochem, 21

Morrow, G.; Inaguma, Y.; Kato, K.; T anguay, R. (2000) T

protein Hsp22 of Drosophila melanogaster is a M

Displaying Oligomeric Organization. J Biol Chem, 275:31

Tanguay, R.; Joanisse, D.; Inaguma, Y.; Michaud, S. (19

proteins: in search fro functions in vivo. In: Storey KB

Stress and Gene Regulation. BIOS Scientific Publishers

138.

ProcedureProcedureProcedureProcedure

HSP2

2(bp)cDNA

HS

P22(bp)cD

NA

pET30-HSP22pET30-HSP22 pET30-HSP27pET30-HSP27

HSP27(

bp)cDNA

HSP27(bp)cDNA

Protein expression inductionProtein expression inductionProtein expression inductionProtein expression inductionProbing with -hsp27 antibody Probing with -hsp22 antibody

+ - + + - - M + - + + - - G J1 1 5 8E s t i rp e s E .

- + - - + + M - + - - + + B L 2 1 D E 3

- + - + - + M - + - + - + H S P 2 2V e c t o r p E T

+ - + - + - M + - + - + - H S P 27

+ - + + - - M + - + + - - N a C l I nd u o

- + - - + + M - + - - + + I PT G

hsp27hsp27 hsp22hsp22

celllysate

Flowthrough

Washwith

BB

Washwith

WB

Elution

Flowthrough

Introduction

Materials and MethodsMaterials and Methods

ReferencesReferences