Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization

MaryAnn _{Liebert, Inc.,} Publishers

Pp. 429-436

Production

of

Human

Recombinant

Proapolipoprotein

A-I

in Escherichia coli:

Purification

and Biochemical Characterization

NICOLE

_{MOGUILEVSKY,*}

CORNELIS

ROOBOL.t

ROSETTE

_LORIAU,*

JEAN-PAUL

GUILLAUME,*

PAUL

_JACOBS,*

ALFREDO

CRAVADOR,*

ALBERT

_HERZOG,*

LÉON

BROUWERS.t

ALAIN

SCARSO.t

PASCAL

GILLES.t

LEIF

_HOLMQUIST.t

LARS A.

_CARLSON.Î

and ALEX BOLLEN*

ABSTRACT

A human liver cDNA

_library

was usedtoisolateaclone

coding

for

apolipoprotein

A-I

(Apo

A-I).

The clone

carries thesequence forthe

prepeptide (18

amino

acids),

the

_propeptide

₍₆

amino

acids),

and themature pro-tein

₍₂₄₃

amino

acids).

A

_coding

cassette forthe proapo A-I moleculewas reconstructed

by

fusing

synthetic

sequences,chosento

optimize expression

and

_specifying

the amino-terminal methionine and aminoacids -6

to

_+14,

to a

_{large fragment}

of the cDNA

_coding

for amino acids 15-243. The module was

expressed

in

pOTS-Nco,

anEscherichia coli

expression

vector

_carrying

the

_regulatable

X

_P^

_promoter,

_leading

to the

pro-duction of

_{proapolipoprotein}

_{A-I at up} to 10% oftotal soluble

proteins.

The recombinant

polypeptide

was

purified

and characterizedintermsof

_apparent

molecularmass, isoelectric

point,

and

_by

bothchemical and

enzymatic

peptide

_mapping.

In

_addition,

it was

assayed

in vitro for the stimulation ofthe enzyme lecithin:

cholesterol

_{acyltransferase.}

The data show

for

the first time thatproapo A-Icanbe

produced efficiently

in

E. colias a stable and

undegraded protein

having

physical

and functional

properties

indistinguishable

from

those ofthe natural

product.

INTRODUCTION is

_responsible

for the formationofmost ofthe

_cholesteryl

estersin

_plasma

_(Fielding

et

al,

1972).

Apo

A-I and HDL

Human

apolipoprotein A-I

(apo A-I),

the

_major

pro- may

participate

in "reverse cholesterol

transport,"

from

pe-tein constituent of

_{high-density lipoproteins}

(HDL),

ripheral

tissues to the liver for excretion from the

_body

is

_produced

in liver and intestine as a _precursor

protein

(Glomset, 1968).

(preproapo A-I)

which

_undergoes

intracellular

cleavage

Since accumulation of cholesterol in the arteries is the

andis released into the

_plasma

and

_lymph

asa

_proprotein

hallmark and

presumably

an

_important

cause for

athero-(Wu

and

Windmueller,

1979).

Proapo

A-Icarries six addi-

sclerosis,

stimulation of reverse cholesterol transport

by

tional amino acids

_{(Arg-His-Phe-Trp-Gln-Gln)}

at the

_{supplementing}

_{apo A-I}

_{might delay}

the atherosclerotic amino-terminalof the mature

_protein.

It is cleaved inthe _process.

_Techniques

to

produce

this

_protein

in

_large

quan-vascular _space

by

a

_specific

_protease to

_yield

the mature tities have been

developed

using

blood

_plasma

as

starting

molecule

_(Gordon

et

al, 1983;

Zannis et

al,

1983).

Apo

material

(Ross

and

Carson,

1985; Breweret

_{al, 1986).}

At-A-I is a

single

nonglycosylated polypeptide

ofknown se- tempts to_expressmature_apoA-I in recombinant bacteria

quence,

composed

of 243 amino acid residues

(Brewer

et

proved

difficult due to the

_instability

of the

protein

ai, 1978).

Inthe_presenceof

_{synthetic liposomes,}

it serves

_(Lorenzetti

et

al,

1986;

Mallory

et

_{al., 1987);}

moreover,

as cofactor for lecithin ¡cholesterol

acyltransferase,

which even as afusion

protein,

_apoA-I wasstill_verysensitiveto

•Service de_{Génétique Appliquée,} Université Libre deBruxelles, 24rue del'Industrie, B-1400 Nivelles, Belgium.

tu.C.B.

and U.C.B. Bioproducts,chemin du Foriest, B-1420 Braine-l'AUeud,_Belgium.

ÍKing

GustafVResearch Institute, KarolinskaInstitute, Box60004, S-10400Stockholm, Sweden.

degradation

and could not be cleaved

_efficiently

from the

fusion

(Lorenzetti

et

_{al., 1986;}

Monaco et

_al,

1987).

To

circumvent these

_problems,

we

_expressed

_apo A-I as the

proprotein.

We show here that proapo A-I can _be

pro-duced

_efficiently

in Escherichiacolias astable and

unde-graded molecule,

having physical

andfunctional

_properties

identical to those of the natural

_product.

MATERIALS AND METHODS

Plasmids, strains,

and media

Three

_{plasmids, pBR322 (Bolivar}

et

_al.,

_1977),

pULB1221

(a

multi-cloning

site derivative of

_pULB1219,

Cravador et

al., 1985),

_pOTS-Nco

(Devare

et

al,

1984),

and three E. coli

strains,

MM294

(Lawn

et

_al.,

_1981),

AR58,

and AR120

(Mott

et

al,

1985),

were used. M33

minimal medium is described

_{by Fujimoto}

et al.

_(1988).

RNA

preparation; synthesis,

cloning,

screening,

and

sequencing of

cDNA

Proceduresto_prepareandscreenthe humanlivercDNA

library

were as described before

_(Jacobs

et

al,

1985).

Oligonucleotides

were

synthesized

by

the

phosphoramidite

method on an

_{Applied Biosystem}

Synthesizer,

model380A

(Matteucci

and

_{Caruthers, 1981).}

_{DNA sequence}

_analysis

was

performed by

the methods of Maxam and Gilbert

(1977)

and

Sanger

et al.

(1977).

Immunological

detection

_of

proapoA-I in bacterialextracts

Transformed bacterial strains were _grown in 20 ml of

rich

_(LB)

or minimal medium

_supplemented

with

ampicil-lin

(50

/ig/ml)

to an _OD630 of 0.6. Induction of the X

_PL

promoter was achieved either

by

_shifting

the temperature from30°Cto

_42°C,

or

by

adding

nalidixic acid

(60

fig/ml)

to cells grown at 37°C

(Mott

et

al., 1985).

Induction was

performed

forvarious

_periods

of time as indicated in the

figure legends.

One-milliliter

_aliquots

of cultures were collected and

centrifuged

at

10,000

x _g for 5 min. Pellets were

resus-pended

in 50

_¡A

ofNaDodS04

sample

buffer

₍₅₀

mM Tris

HC1

pH

6.8

_containing

2%

NaDodS04,

6 M_urea, 5%

2-mercaptoethanol,

and 10%

glycerol),

boiled for 3 minat

100°C,

and

centrifuged

for 10 minat 15,000 x _g.

_Samples

werethen fractionated

by electrophoresis

on 12.5or7.5%

NaDodS04-polyacrylamide gels

as described

_by

Laemmli

(1970).

The

_proteins

wereblotted ontonitrocellulose sheetsand tested for the _presence of proapo A-I with antibodies raised

_{against plasma}

_{apo A-I.}The

_procedure

follows Bol-len et al.

(1984),

except that the second

_antibody,

goat

antimouse,

was labeled with alkaline

_phosphatase

instead

of

_peroxidase,

and that the

_chromogenic

substrates were

BCIP

_{(5-bromo-4-chloro-3-indolyl-phosphate, Boehringer)}

and NBT

(4-nitrotetrazolium-chloride blue, Sigma),

dis-solved in 100 mM Tris-HCl

pH 9.5,

100 mM

NaCl,

and 50 mM

_MgCl2

at 66and 33

_{fig/ml, respectively.}

Purification of

recombinantproapoA-I

Packed bacterial cells

_(usually

35

grams),

derived from 15-liter fermentations in M33

medium,

were

resuspended

intwovolumes ofice-cold extractionbuffer

(20

mM

Tris-HCl

pH

7.5, 100mM

NaCl,

1

mMEDTA,

1 mM

PMSF,

and 100

_{¡ig/ml merthiolate)}

and

_{disrupted by}

a

_single

pas-sage

through

a French's_pressure cell at _1,000 PSIG. The

extractwas

_centrifuged

for 15 min at

4,000

x _gto

sedi-ment membranes and _aggregates then

_applied

onto a

Phenyl Sepharose

column CL-4B

(bed volume,

25

ml;

sample,

100

ml).

The column wasthen

sequentially

eluted

with 250 mlof extraction

buffer,

250 mlof thesamebuffer

supplemented

with 60%

_{(vol/vol) propylene glycol,}

and 250 ml of thesamebuffer without NaCl and

supplemented

with80%

_{(vol/vol) propylene glycol.}

Fractions

_containing

proapo A-Iwere

pooled

and then

applied

onto a PBE 74

chromatofocusing

column

(0.8

x 60

cm),

which was

elutedwithtwobed volumes ofabuffer

containing

25 mM

imidazole-HCl

pH

6.0 and 6 Murea, followed

by

10 bed

volumes of

_polybuffer

74

_(dilution

_{1/10) pH 4.0,}

6 Murea. The columnwas

regenerated by washing

with 50 mM

Na-citrate

_pH

4.0,

1 M

NaCl,

and 6Murea. Fractions were

assayed

for total

protein

content and _apoA-I-related

im-munoreactivity,

and

analyzed by

isoelectric

_focusing.

Chemical

peptide mapping

with BNPS-Skatol

Chemical

_cleavage

with BNPS-Skatolwas

performed

ac-cording

to the

_procedure

ofFontana

(1972). Briefly,

5-10 fig of

protein

were dissolved in 100

¡A

of a solution of

0.15%

_{(vol/vol) phenol}

in50%

(vol/vol)

acetic acid.

Then,

50

fA

ofasolutionof 4.8_mgBNPS-Skatol_permilliliter of

glacial

acetic acid were

added,

followed

by

an incubation

of 72 hr at25°C.

_{Subsequently,}

50

_¡A

of

2-mercaptoetha-nol was

added,

followed

by

a second incubationof 5 h at

37°C. The

_samples

were

_evaporated,

redissolved in 100

_fA

of water, andextractedthree times with 200

_^tl

of

ethyl

ace-tate.The

_organic

phases

were

discarded,

whereas the aque-ous

_phases

were

lyophilized

and

analyzed

by

acrylamide

gel electrophoresis.

Stimulation

_of

lecithin:cholesterol

_{acyltransferase}

activity by

recombinantproapo A-I

Human

_plasma

apo A-I was

purified according

to

Holmquist

and Carlson

(1977)

and

_Holmquist

and

Bro-strom

(1979).

Simple

bilayer

vesicles

_containing

_egg leci-thinand

_{7(/i)-3H-labeled}

cholesterol

(molar

ratio

4:1)

were

purified

human

_plasma

lecithin:cholesterol

_{acyltransferase}

(35,000

units/mg)

was

prepared

_according

to

_Holmquist

(1987).

Incubations and enzyme

activity

determinations

werecarried outin 50 mM

_phosphate

buffer

_pH

7.4as

de-scribed

by

Piran and Morin

(1979).

Incubation mixtures

(250

fA)

contained _perliter:240

_/¿moles

lecithin,

60

_/¿moles

cholesterol,

and 3 or 6

/¿moles apolipoprotein.

Miscellaneous

_procedures

Protein concentrationsweredetermined

according

tothe

procedure

of Bradford

(1976),

using

ovalbumin asa

stan-dard. Partial

_tryptic

hydrolysis

was

performed

_according

tothe

_procedure

ofAmonsetal.

(1983).

Human apo A-I-related

_{immunoreactivity}

was

_quantified

_by

radial

immu-nodiffusion

(Cheung

and

_{Albers, 1977),}

_using

commercial

apo A-I

(Sigma)

as astandard. The relative

immunoreac-tivity profile

were determined

by

immunoturbidimetry

(Shapiro

et

_{al, 1980).}

Isoelectric

_focusing

was carriedout

according

to the

_procedure

of

Catsimpoolas (1968)

in a

Bioradrod

_{electrophoresis}

apparatus,

applying

a

self-gen-erating gradient

from

pH

4to

pH

6. Linear and nonlinear

regression analyses

were

performed

withtheStatView

pro-gram

(Brain

Power Inc.,

USA).

RESULTS

Molecular

_{cloning of}

_Apo

A-IcDNA and

_screening

of

the cDNA

_library

Total

_poly(A)*RNA

(25

fig)

fromhuman liverwas

tran-scribed into double-strandedcDNA

_(ds

cDNA).

Molecules

resistant to S, nuclease were then fractionated on a

10-30% sucrose

gradient

to enrich for molecules

_larger

than 0.8 kb. This ds cDNA fractionwasinserted into thePst I

siteof

pBR322 by

thedC:dG

_tailing

procedure

andusedto

transform E. coli MM294 cells. The

_library

was screened

by

the

colony hybridization

method

using

a 22-base

syn-thetic

_{oligonucleotide probe}

derivedfromthesequence for apo A-I

(Cheung

and

Chan, 1983)

and

coding

for amino

acids -22 to -15 ofthe

signal

peptide.

Of the 30 clones

hybridizing

with the labeled

probe,

one,

pNIV1602,

was

chosen for further

_analysis.

Characterization

of

theclone

_pNIV1602

Clone

_pNIV1602

carriedaninsertof 878

bp

whichcould

be isolated as a

_{single fragment by digesting}

the

_plasmid

with Pst I.The insert was

mapped

with variousrestriction

enzymesand

fully sequenced

oneach strand. Thedata

(not

shown)

showed that

pNIV1602

carries

(i)

a

_19-bp

5'

non-coding

sequence,

(ii)

a

75-bp

_sequence

_{corresponding}

to

the known

signal

and

propeptide

region

of apo

A-I,

in-cluding

the ATGinitiation

codon,

(iii)

the

_{complete 729-bp}

sequence

corresponding

tothemature

_protein,

followed

by

aTGAstop

codon,

and

_(iv)

a

55-bp

3'

noncoding

_sequence

that did notcontain the

_poly(A)

stretch. The _{sequence of} thecDNAinsertwasidenticaltothesequence

published

by

Cheung

and Chan

(1983),

but differed_{from the sequence}

of Seilhameretal.

(1984)

by

afew bases and

by

the

length

of the 5' and 3'

noncoding regions.

The deduced amino

acid _sequence,

however,

wasconsistentwith the

_published

data

_(Cheung

and

Chan, 1983;

Seilhamer et

al.,

1984).

Construction

_of

the recombinant

_plasmid

pNIVl617

Theconstruction

_proceeded

in twosteps. Thefirst used

the

_{multi-cloning}

site

_plasmid

pULB1221,

linearized with

Bal

I,

as anintermediatevehicletosubcloneablunt-ended

744-bp

Bal l-Pst I DNA

_fragment

derivedfrom the clone

pNIV1602

and

_coding

for amino acids 15-243 ofthe _apo

A-I

protein.

The

_{resulting plasmid}

was

digested

with Eco RI and Bal I and fused to

_(i)

the

1,880-bp

Eco Rl-Nco I DNA

fragment

containing

the

_{regulatory regions}

derived

fromthe

_expression

vector

_{pOTS-Nco (Devare}

et

_{al., 1984)}

and

(ii)

a

_65/61-bp

Nco l-Bal I

synthetic

DNA

_adaptor

en-coding

theamino-terminal methionineand amino acids -6

to _{14 of proapo A-I}

_(Fig.

_1).

The

resulting

recombinant

plasmid,

pNIV1617,

thus carried the _strong

_regulatable

X

Pl

promoter upstreamfromthecassette

_coding

for proapo A-I.

The

synthetic

adaptor

wasassembled fromfour

oligonu-cleotides

_{(Fig. 2)}

whose base _sequence was

designed by

computertominimize

_secondary

structuresatthe5' endof

the

_coding

_sequence

(S.A.S.I.P.

software

_package;

Clav-erie,

1984). Triplets

encoding

aminoacidresidues -6, -1, 1,

3, 4,

5, 6,

7, 10, 11,

and

14,

therefore differed fromthe natural ones

(Table 1),

_although

the

_resulting

amino acid

sequence was

_preserved.

Plasmid

pNIV1617

wascharacterized

by

restriction

anal-ysis,

hybridization

with labeled

synthetic

DNA

_fragments,

and

_{sequenced throughout}

the

adaptor

region.

Itwasthen

introduced into E. coliAR58

_(for

thermal

_induction)

and

AR120

(for

chemical

_induction).

Expression

of

proapoA-I

Cells

_carrying

either the control

_plasmid,

_pOTS-Nco,

or

the

pNIV1617 plasmid

were_grownat30°C inminimal

me-dium,

for thermal

induction,

or at 37°C in rich

medium,

forchemical induction.

_Derepression

of the X

_PL

promoter

wasachievedeither

by

shifting

thetemperatureto42°Cor

by

adding

nalidixic acid. Inductionwas

performed

for

var-ious

periods

of time as indicated in the

legend

to

_Fig.

3.

Pelletedbacteriawere

lysed

andthe

_resulting

extractswere

analyzed

by

NaDodS04-polyacrylamide gel

electrophore-sis,

Western

_blotting,

andimmunodetection

_using

antibod-ies raised

_{against plasma}

_{apo A-I.}

Figure

3 shows that a

single

product, specifically

recognized by

anti-apo

A-I

antibodies,

is

expressed

in the bacterial strains

_carrying

plasmid

pNIV1617,

irrespective

ofthe modeofinduction.

apoA-l BalI "15 synthetic DNA ATG Ncol Met.aa_ T40NA ligase Antpr

Ball-EcoR Ifragment

13 07kbl

Amp

EcoRl-Nco Ifragment

(1 BBkb)

proapoA-l

"»-6 -aa243'

The

_protein

hadamolecularmassofabout

28,000

daltons

which,

within

_experimental

limits,

fittedthecalculated

mo-lecular

_weight

of_{proapo A-I.}

Levels of proapo A-I

produced

in bacteria were

esti-mated

by

single

radial immunodiffusion. Under thermal

induction conditionsin M33 minimal

medium,

proapo A-I

was

_expressed

at about 10% of total soluble

proteins

versus 3.5% inLB medium. Chemical induction

_appeared

tobe less

_{efficient, yielding}

_expression

levels on theorder

of2% in LB mediumand no

_expression

in M33 medium.

Purification of

recombinantproapo A-I

We used

_hydrophobic

interaction

_{chromatography}

to

purify

the recombinant _{proapo A-I.} This

approach

al-lowed an excellent

separation

of recombinant molecules

from otherbacterial constituents

_(Fig.

4,

peak

III).

Chro-matofocusing

on aPBE-74 columnwasusedto

_purify

the

product

further. As shown in

_Fig.

5, immunoreactive

ma-FIG. 1. Construction of

_plasmid

pNIV1617.

A

_744-bp

Bal l-PstI

fragment

derivedfromclone

pNIV1602,

coding

for amino acids 15-243 of apo

A-I,

was

_ligated

into the

unique

Bal I site of

pULB1221.

From the

_resulting

plas-mid,

the

_large

Eco Rl-Bal I

_fragment

was fused to the

1,880-bp

Eco Rl-Nco I

_fragment

derived from

pOTS-Nco

andtothe

synthetic

adaptor

Nco l-Bal Itoregeneratea

se-quence

coding

for proapo A-I.

Hybrid

molecules were

usedto transformE. colicellsandtransformantswere

se-lected for

_ampicillin

resistance. The nucleotide _{sequence of} the

_junctions

between

_fragments

and ofthe full

synthetic

fragment

were determined.

Ncol site

5-- -35 mer

BalIsite

-3'5' -30

mer-CATG AGA CATTTCTGG CAG CAG GAC GAA CCT CCA C AA TCT CCTTGG GATAGA GTTAAGGAC TTG G TCT GTA AAGACC GTC GTC CTG CTTGGA GGT G TT AGAGGAACC CTATCT CAATTCCTGAAC C

3"-18 met--53-"- 43 _mer-»5

Met _Arg His Phe Trp Gin Gin

6 -3 1

NH-, terminal propeptide

methionine " ₆ aa

1 '

Asp Glu Pro Pro Gin Ser Pro Trp Asp Arg Val Lys Asp Leu A(la)

»1 +3 +6 +9 +12 ₊₁₄

mature apo A-l (aa.

-"14

cleavage site for _apoA I_propeptidase

FIG. 2.

_{Synthetic adaptor}

usedtoreconstruct _{the proapo A-I} cDNA. Four

_oligomers

were

synthesized

_andassembled

in vitrotogeneratea

_65/61-bp

_fragment

flanked

_by

aNcoIandaBal Isitesonthe5'and3'

ends,

_{respectively.}

The de-duced aminoacid_sequenceisindicated. The arrowshowsthe known

_cleavage

site for_{apo A-I}

_propeptidase

_(Gordon

et

Table 1. Relevant Changesin CodonsUsed toReconstruct

theProapo A-I cDNA Position in

the

protein

Aminoacid Naturalcodon

Modified

codon

-6 -1 1 2 3 and4 5 6 7 8 9 10 11 12 13 14

Arg

Gin

Asp

Glu Pro Gin Ser Pro

Trp

Asp

Arg

Val

Lys

Asp

Leu CGG CAÁ GAT GAA CCC CAG AGC CCC TGG GAT CGA GTG AAG GAC CTG AGA CAG GAC CCC and CCA CAA TCT CCT AGA GTT TTG

Modifications in the codon usageweredone in ordertominimizesecondary

structures in the 5' end of the proapo A-I cDNA. The software package

S.A.S.I.P.developed bythe Institut Pasteur_(Paris)wasused(Claverie,1984).

11 10 987 65432 1 kDa

A

.43 13 12 11 10 9 8 7 6 5 4 3 2 1 kDa

B

_29 .18.4 29 .18.4

FIG. 3. Immunodetection _{of proapo} A-I on Western blots. Protein extracts from induced and noninduced bacteria

carrying

plasmid pNIV1617

were

_prepared

for

_{electrophoresis}

on

NaDodS04-polyacrylamide gels.

Nitrocellulose blots wereincubated withmouseanti_apoA-Iserum, followed

by

alkaline

phosphatase-labeled

goatanti-mouse and the

chro-mogenic

substrate. A. Timecourseof thermalinduction ofbacteria

carrying

pNIV1617

and growninM33minimal

me-dium. Lane

1,

Plasma derived _apoA-I, 500 _ng; lanes

2-6,

samples

takenat 1,

20, 40, 60,

and 120 minin noninduction conditions

_(30°C);

lanes 7-11,

samples

taken at

1, 20,

40,

60,

and 120 minafter induction

(42°C).

B. Time courseof

chemicalinduction

_{(nalidixic acid)}

ofbacteria

_carrying

pNIV1617

and_growninrichLBmedium.Lane 1,Plasma derived apo

A-I,

500 ng; lanes

2-7,

samples

taken at

1, 30, 60, 120, 180,

and240minin noninduction conditions

(37°C);

lanes

terial

_{(fractions 37-48)}

was resolved from contaminants

andwasidentified

_by

isoelectric

_focusing

mainly

as_proapo

A-I.

Moreover,

chromatofocusing

resolved most _{apo A-I}

relatedisoforms.

Characterization

of

purified

recombinant

proapoA-I

Pure recombinant _proapo A-I was

analyzed by

NaDodS04-polyacrylamide

gel

electrophoresis

and isoelec-tric

_focusing.

_{Recombinant proapo A-I}behavedasa

_single

Material Eluted. Arbitrary Units

h.

I

Ccccccccctíí?

<ccccCrccCco°co_

20 30 FractionNumber -•rft>-40

FIG. 4.

_Hydrophobie

interaction

_{chromatography.}

A total of 100 ml ofclearedextractof bacterial cells derived

from 15-liter fermentation in M33 medium was

_subjected

to

_hydrophobic

interaction

chromatography

as described

inMaterials and Methods. The eluatewas

_assayed

fortotal

protein (O)

and apo A-I-related

immunoreactivity

(•).

PeakIIIcontains the

_majority

of therecombinant

protein.

polypeptide

chain with an apparent molecular mass of

(27.3 ±1.1)

kD.Matureapo A-Iwas

_slightly

smaller,

hav-ing

an _{apparent molecular}

_weight

of

_(26.8

±

1.1)

kD. In

isoelectric

focusing (data

not

shown),

the recombinant

protein

showedone

_major

isoformandoneminor

isoform,

having

isoelectric

_points

of 5.20 and

4.95,

respectively.

The

relative

_migration

distances of these isoforms coincided

exactly

with those of

_plasma

proapo A-I andapo

A-I,

re-spectively.

Partial chemical

_cleavage

at

_tryptophan

resi-dues,

was

performed

on recombinant _proapo A-I

_(major

isoform)

and on

plasma-derived

_{apo A-I.}

_Figure

6 shows

the

_{large peptides}

of

expected

molecular

_{weight produced}

in both cases

_{by cleavage}

at

_positions

8, 50, 72,

and 108 in

the

_proteins.

In

addition,

digestions

of recombinant

proapo A-I and of

plasma-derived

apo A-I with

trypsin

in

verymild conditions

yielded

similarpatterns of

fragments

(data

not

_shown).

_Thus,

_{both types of}

_experiments

suggest that the recombinant molecule is

_structurally

identical to

its natural _counterpart.

Functional

_analysis

of

recombinantproapoA-I

Recombinant proapo A-I

(pi 5.20)

and its minor iso-form

(pi 4.95)

were both ableto transform

synthetic

leci-thin-cholsterol

_liposomes

into substrates for the _enzyme

lecithin:cholesterol

_{acyltransferase.}

AsseeninTable

2,

the

activation

_capacity

of recombinant

_apoproteins

wassimilar

totheoneobservedwithauthentic human

_plasma

_{apo A-I.}

kDa

_J

Material Eluted, Arbitrary Units T-6 +5 30 40 50 Fraction Number

FIG. 5.

_{Phenyl-sepharose chromatography.}

Peak III of the

_{phenyl sepharose}

eluate was

_subjected

to

chromatofo-cusing

as described in Materials and Methods. The eluate

was

assayed

for total

protein (O),

_apoA-I-related

immu-noreactivity (•),

and

pH

(A).

Fractions 37-48 contain the

purified

recombinant

product.

43

30

20.1"

****'**

14-4-FIG. 6. Chemical and

enzymatic

characterization_of

pro-apo A-I. Purified recombinant proapo A-I and human

plasma

apoA-I were

_subjected

to

partial

chemical

_peptide

mapping

as described in Materials and Methods. Lane

1,

Molecularmass standards

_(Pharmacia,

14,400-94,000

dal-tons)

indicated

_by

_arrows; lane

2,

human

_plasma

_apo

_A-I,

untreated;

lane

3,

human

_plasma

_apo

A-I, cleaved;

lane4,

recombinant _proapo

A-I, untreated;

lane

5,

recombinant

Table 2. Stimulation of Lecithin ¡Cholesterol Acyltransferase Activity by

Recombinant Proapo A-I Source

LCAT

_{Apolipoprotein}

LCAT

_activity

(nmoles/hr

x

_liters)

Plasma apo A-I

Recombinant _{proapo A-I}

Major

isoform

(pi 5.20)

Minor isoform

(pi 4.95)

25 25 100 100 100 100 100 100 0 3 0 3 6 0 3 6 2.5 250 100 850

1,600

100 700 700

Synthetic liposomes (lecithin/cholesterol, 4:1)wereincubatedat37°C for 2-4hr,inatotal vol-umeof 250(A,with LCAT(25units/ml)and variousamountsofapoA-I. The initialrateof

chole-sterylesterformation _{(LCAT activity)}wasmeasuredaccordingtoPiran and Morin_(1979).

DISCUSSION

Wehave cloned a cDNA

_coding

for human _preproapo

A-I;

its deduced amino acid sequence fits the

published

data

(Cheung

and

Chan, 1983;

Seilhameret

al.,

1984)

and

is almost identicaltothat

_reported

by

Breweretal.

_(1978).

Weconstructedamodule

_coding

forthe_proapoA-I mole-cule and

_expressed

it in E. colt under the control of the

regulatable

X

_PL

promoter.

Proapo

A-I,

produced

at

_high

level,

was

stable,

_{largely undegraded,}

and

_{indistinguishable}

from the natural

product

in terms of_apparent molecular

mass,isoelectric

point,

and

partial

peptide

map. As

expected,

mostof the material

produced

in E. coliwasindie

propro-tein

form,

_although

the_presenceofanamino-terminal

me-thioninecannot be excluded yet. The

_purified

protein

ap

peared

to contain a minor isoform which coincided with

mature _{apo A-I}

_by

isoelectric

focusing. However,

it is

un-certain if this is duetodeamidationortoconversion ofthe recombinant

_protein

to mature_{apo A-I.} The

_high

level of

expression

observed for _{proapo A-I in}bacteriacontrasted

with the relative failure

_(Lorenzetti

et

al, 1986;

our

data,

not

_shown)

to achieve

_{significant expression}

of a

stable,

undegraded

mature _{apo A-I}

_species

in the samehost. The

dramatically

increased

_expression

associatedwith the

pres-enceofthe

_propeptide

sequence

might

be

explained

in

sev-eral _ways.

Transcriptional

or translational efficiencies

might

be increased due to the extra _sequence.

Alterna-tively,

the

proprotein species

might

fold more

stably

in

bacteria thanthe mature

protein

andhencebemore

resis-tant to

_degradation.

This last

_hypothesis

seems more

probable

since

_pulse-chase

experiments

have shown that thehalf-life ofmature _{apo A-I in}bacteria is less than 10

min

(Mallory

et

_{al, 1987).}

Our

data,

in

addition,

offer

di-rect evidence for the first time that _{recombinant proapo}

A-I is able to stimulate in vitro the

enzymatic

activity

of lecithin:cholesterol

acyltransferase,

as

_efficiently

as

plasma-derived

apoA-I. In this respect,proapo A-I

might

constitute an

appropriate

substituteto mature_{apo A-I} for

replacement

therapy, provided

that

_{pharmacological}

stud-ies in animal models confirm its functional

_efficiency.

ACKNOWLEDGMENTS

This work was

_performed

under a contract between U.C.B. and the

_University

ofBrussels and

supported by

a

grant from the Swedish Medical Research Council

(19X-04)

The authorsthank Drs. A. Shatzmanand M. Rosen-L i

¡Smith

Klineand

French,

Philadelphia,

PA)

for

pro-viding

the

pOTS-Nco

vector and the E. coli AR58 and AR120strains.

REFERENCES

AMONS, R., PLUYMS, W., ROOBOL, C,andMOLLER, W.

(1983). _Sequence homology between _{EF-la, the a-chain of}

elongationfactor1 from Artemia salina and_elongation factor EF-Tu from Escherichia coli. FEBS Lett. 153,37-42.

BATZRI, S.,andKORN,E.(1973).Single bilayer liposomes

pre-pared without sonication. Biochim. Biophys. Acta298, 1015-1019.

BOLIVAR, F., RODRIGUEZ, R.L., GREENE, P.J.,

BET-LACH, M.C., HEYNECKER, H.L., BOYER, H.W., CROSA, J.H., and FALKOW, S. _(1977). Construction and

characterization of new cloning vehicles. IL A multipurpose cloning system. Gene2,95-113.

BOLLEN, A., LORIAU, R., HERZOG, A., and

HÉRION,

P.

(1984). Expressionof humana,-antitrypsininEscherichiacoli. FEBS Lett. 166,67-70.

BRADFORD,M.M. (1976).Arapidand sensitivemethodfor the

quantitation of _{microgram quantities} of _{protein utilizing the}

principleof_{protein-dyebinding.}Ann. Biochem. _72, 248-254.

BREWER, H.B., FAIRWELL, T., LARUE, A., ROÑAN, R., HOUSER,A.,andBRONZERT,T._(1978).The amino acid

se-quence of human apo A-I, an apolipoprotein isolated from

high density lipoproteins. Biochem. Biophys. Res. Commun.

80, 623-630.

BREWER, H.B., RONAN, R., MENG, M., and BISHOP, C.

(1986). Isolation and characterization ofapolipoproteinsA-I,

A-II and A-IV. MethodsEnzymol. 128,223-246.

CATSIMPOOLAS, N. _{(1968). Microisoelectrofocusing} in

poly-acrylamidegels columns. Ann. Biochem.26,480-482.

CHEUNG, M.C., and ALBERS, J.J. _{(1977). The} measurement

ofapolipoproteinA-IandA-IIlevels inmenandwomenby im-munoassay. J. Clin. Invest. 60,43-50.

CHEUNG, P., and CHAN, L. _(1983). Nucleotide sequence of

clonedcDNA of human_{apolipoprotein}A-I.NucleicAcids Res.

11, 3703-3715.

CLAVERIE, J.-M. _(1984). A common philosophy and

FOR-TRAN77software_packageforimplementingandsearching

se-quence databases. Nucleic Acids Res. 12, 397-407.

CRAVADOR, A., JACOBS, P., VAN_{ELSEN, A., LACROIX,}

C, COLAU, B., VAN ALPHEN, P., HERZOG, A., and

BOLLEN, A. _(1985). Total DNA _synthesis and_cloning in E. coli ofa_genecodingfor the human Growth Hormone

Releas-ing Factor. Biochimie67, 829-834.

DEVARE, S.G., SHATZMAN, A., ROBBINS, K.C.,

ROSEN-BERG, M.,andAARONSON,S.(1984)._Expressionof

PDGF-relatedtransforming proteinof simiansarcomavirus in E. coli. Cell36,43-49.

FIELDING, C.J., SHORE, V.G.,and_FIELDING,P.D._(1972). A_proteincofactor of lecithin: Cholesterolacyltransferase. Bio-chem. _Biophys. Res. Commun. 46, 1493-1498.

FONTANA, A. _(1972).Modification oftryptophanwith BNPS-skatol. MethodsEnzymol.25, 419-423.

FUJIMOTO, S., NAKATSU, M., KATO, K.,andKITANO,K.

(1988).Factors_affectingthesynthesisof the N-terminal methi-onine-free molecule of recombinant human interleukin-2 _by Escherichia coli. J. Ferment. Technol. 66, 181-185.

GLOMSET, J.A. _(1968). The _{plasma lecithin: Cholesterol} acyl-transferase reaction. J. _Lipid Res.9, 155-167.

GORDON, J.I., SIMS, H., LENTZ, S.R., EDELSTEIN, C, SCANU, A.M., andSTRAUSS,A.W.(1983). _Proteolytic

pro-cessing of human preproapolipoprotein A-I. J. Biol. Chem.

258,4037-4044.

HOLMQUIST, L., and_CARLSON, K._(1977). Selective

extrac-tion of humanserumverylowdensityapolipoproteinswith

or-ganicsolvents. Biochim. Biophys. Acta493,400-409.

HOLMQUIST, L.,andBROSTROM, H. (1979).A_simple

Beck-manDUaccessoryfor location ofproteinsseparatedby isoelec-tric_focusingin_{granulated gel layers:}Isolation of humanserum

verylow densityapolipoproteins C-II and C-III. J. Biochem.

Biophys. Methods 1, 117-127.

HOLMQUIST, L.(1987).Purification of humanplasmalecithin:

cholesterol _{acyltransferase} by covalent _{chromatography.} J. Biochem. Biophys. Methods 14,323-333.

JACOBS,P., CRAVADOR, A., LORIAU, R., BROCKLY, F., COLAU, B., CHUCHANA,P.,VAN_{ELSEN, A.,}HERZOG, A., and BOLLEN, A. (1985). Molecularcloning, sequencing

and _expressionin Escherichia coli of human _{preprourokinase} cDNA. DNA 4, 139-146.

LAEMMLI, U.K.(1970). _Cleavageof structural _{proteins during} the assembly of the head of _{bacteriophage} T4. Nature 227,

680-685.

LAWN, R.M., ADELMAN, J., BOCK, S.C., FRANKE, A.E., HOUCH, CM., NAJARÍAN, R.O., SEEBURG, P.H., and

WION, K.L. (1981). The sequence of human serum albumin cDNA and itsexpressioninE. coli. Nucleic Acids Res.9, 6103-6114.

LORENZETTI, R., SIDOLI, R., PALOMBA, R., MONACO, L., MARTINEAU, D., LAPPI, D.A.,andSORIA, M._(1986).

Expressionof the humanapolipoproteinA-I genefusedtothe E. coli_genefor_{/3-galactosidase.} FEBS Lett. 194, 343-346.

MALLORY, J.B., KUSHNER, P.J., PROTTER, A.A., COFER, CL., APPLEBY, V.L., LAU, K., SCHILLING, J.W., and VIGNE, J.-L. (1987). Expressionand characteriza-tion of human _{apolipoprotein} A-I in chínese hamster _ovary

cells. J. Biol. Chem. 262, 4241-4247.

MATTEUCCI, M.D.,andCARUTHERS, M.H.(1981).

Synthe-sis of _{deoxyoligonucleotides} on a polymer _support. J. Am.

Chem. Soc. 103,3185-3191.

MAXAM, A.M.,andGILBERT, W. (1977).Anewmethod for

sequencingDNA. Proc. Nati. Acad. Sei. USA 74, 560-564.

MONACO, L., BOND, H.M., HOWELL, K.E.,andCÓRTESE,

R.(1987). Arecombinant apoA-I-proteinA_{hybrid reproduces} the binding parameters of HDL to its _receptor. EMBO J. 6,

3253-3260.

MOTT, J.E., GRANT, R., HO, Y.Z., and _PLATT, T. _(1985).

Maximizing gene expression from _plasmid vectors _containing

the

_PL

_{promoter: Strategies} for overproducing transcription

termination factor P. Proc. Nati. Acad. Sei. USA82, 88-92.

PIRAN, V.,andMORIN,R.J.(1979).A_{rapid radioassay} proce-dure for_plasmalecithimcholesterolacyltransferasebycovalent

chromatography. J. _Lipid Res.20, 1040-1043.

ROSS, S.E., and CASRSON, S.D. (1985). Rapid

Chromato-graphie purificationof_{apolipoproteins}A-IandA-II from

hu-manplasma.Anal. Biochem. 149,166-168.

SANGER, F., NICKLEN, S., and COULSON, A.R. _(1977). DNA_sequencingwith chainterminatinginhibitors. Proc. Nati. Acad. Sei. USA74,5463-5467.

SEILHAMER, J.J., PROTTER, A.A., FROSSARD, P., and

LEVY-WILSON, B. (1984). Isolation and DNA sequence of

full-lengthcDNA and of the entiregenefor human

apolipopro-teinA-I. _Discoveryofa newgenetic polymorphismin theapo A-I_{gene. DNA}3,309-317.

SHAPIRO, D., BALLENTYNE, F.C., and _SHEPHERD, J.

(1980).Comparisonof_{immunonephelometry}and electroimmu-noassay for estimation of plasma apolipoprotein A-I. Clin. Chim. Acta 103,7-13.

WU, A.L.,andWINDMUELLER, H.G._{(1979). Relative} contri-butions_byliver and intestinetoindividual plasma

apolipopro-teins in therat. J. Biol. Chem. 254,7316-7322.

ZANNIS, V.l., KARATHANASIS, S.K., KEUTMANN, T., GOLDBERGER, G., and BRESLOW, J.L. _(1983). Intracellu-lar and extracelluIntracellu-lar _processingof human _{apolipoprotein} A-I: SecretedapolipoproteinA-I_isoprotein2 isapropeptide.Proc.

NatiAcad. Sei.USA80,2574-2578.

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_reprint

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Appliquée

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ruede l'Industrie24 B-1400

Nivelles,

Belgium

Received for_publicationDecember_{27, 1988,}and in revised form