BIOCHEMICAL

Vol. 171, No. 1, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 375-383

August 31, 1990

THE PURIFICATION AND CHARACTERIZATION OF RECOMBINANT HUMAN RENIN EXPRESSED IN TEE HUb¶AN KIDNEY CELL LINE 293 Chris

J. Vlahosl,

Molecular

Received

July

17,

Jenna Brian

Biology

D. Walls, W. Grinnell

Research, Indianapolis,

Lilly

David

T. Berg,

Research IN 46285

and

Laboratories,

1990

The cDNA encoding human preprorenin has been introduced into the adenovirus-transformed human kidney cell line 293. The recombinant 293 cells expressed and secreted prorenin; trypsin was used to activate the secreted prorenin to renin in vitro. The recombinant protein was purified to homogeneity by a single affinity chromatographic step. Using synthetic tetradecapeptide, the Km was 57.1 + 9.3 ulj and the kcat was (7.48 + 1.57) x 103/hr. Activation with trypsin resulted in a secondary cleavage between Arg53 and Leu54 generating a two chain form held together via a disulfide between Cys51 and Cys58. This secondary cleavage did not affect enzyme activity as determined by the ability of renin to degrade a synthetic tetradecapeptide substrate. Our paper demonstrates the potential for producing large quantities of renin from human kidney cells and also suggests that the use of trypsin, which has been widely used to convert prorenin to renin in vitro, causes a secondary cleavage in the renin peptide chain. 01990 Academic

Press,

Inc.

Renin

is

a highly

of angiotensin the

rate

limiting

converted

to

to

mouse for

of

renin

renin

angiotensinogen leucyl-leucine

ITO

catalyzes This

angiotensin

involved

inactive

protease weight

been gland

is

by which

an octapeptide

including

also

that

angiotensinogen.

cascade

as an

molecular

submaxillary

human

human

has

protease

protein

in

the

release

conversion

I

is

vascular

is

ultimately constriction

(1,2).

functional

sources,

Renin

(6).

the

II,

secreted

the

glycoprotein of

in

secretion is

aspartyl

plasma

angiotensin

Renin converted

the

step

and aldosterone

variety

specific

I from

(E),

different

in

from that

a

(lo),

the

scissile

has

stream.

been

plasma

other

for

Human

renin,

from

near

other the

of

then a wide

nonprimate

(7),

specificity

sources. amino

a

chorion

as hog kidney

The cleavage

from

renin

is

and placental such

(9).

which

purified

(5),

sources

uterus bond

bond

(prorenin),

blood

obtained

leucyl-valine

cleaves

Da,

(3,4),

and rabbit from

the

42000

kidney purified

zymogen

Whereas

terminus origins

of is

a

(11).

whom correspondence

and reprint

requests

should

be addressed. OUO6-291x/9o $1.50

375

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 171, No. 1, 1990 Human kidneys is

evidence

the

that

conversion

(14,15). activate only renin

prorenin report

from

also

is

to

occurs

the 293

cells.

during of

cleavage

MATERIALS

kidney

and not

site

in

the but

not

only

the

renin

in the

(12,13); plasma

of recombinant

process

trypsin

and there

in vivo

and purification

secretory with

as renin,

protease

Surprisingly,

the

prorenin

to a secondary

in the

expression

RESEARCH COMMUNICATIONS

as well

by a specific

to renin

renin

prorenin

activated

kidney

Treatment led

to secrete

describes

human

prorenin

prorenin. but

known

of prorenin

This

human renin

are

AND BIOPHYSICAL

293

cells

do not

instead

secrete

produced

active

chain.

AND METHODS

Construction of Expression Vector--Plasmids for the expression of human renin were constructed using the cDNA coding sequence isolated by Faust The renin cDNA clone, inserted into the polylinker of pSP65 et al. (16). was excised on a BamHI to Hind111 restriction fragment and (Promega Biotech), the 3' protruding ends were made blunt by treatment with the Klenow fragment The plasmid phRN-8 (Figure 1) was constructed by of E. coli polymerase. replacing the human protein C coding sequence in pLPC-hd (17) with the blunt ended renin cDNA sequence at the plasmid Bcl.1 site. Cell Lines, DNA Transfection, and Druu Selection--The adenovirus293 (American Type Culture Collection CRL transformed human kidney cell line, 1573), was grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and was transfected with phRN-8 as described previously (18) by the calcium phosphate method (19). Clonal cell lines resistant to These recombinant hygromycin B were isolated as described previously (17). and the presence of renin activity in the cell lines were expanded, conditioned culture medium was determined by the HPLC analysis described below. RNA was isolated from transfected cells and cytodot analysis was performed as previously described (20) using a 32P-labeled probe to the renin cDNA (Hind111 to BamHI restriction fragment). Renin Assay-Renin activity was determined by measuring the simultaneous I and loss of tetradecapeptide substrate using formation of angiotensin isocratic reverse phase HPLC on a DuPont Zorbax ODS column (25 cm x 4.6 mm). A solvent system consisting of 0.1% trifluoroacetic acid in 32% CH3CN was used at a flow rate of 1.5 ml/min. Assay mixtures consisted of 35 run01 renin tetradecapeptide substrate in 100 ul O.lM sodium acetate pH 6.5. Aliquots (20 ul) of column fractions containing renin were incubated at 37OC with the renin substrate. At various times, 20 ul of the reaction mixture was removed, added to 100 ul HPLC running solvent, and 60 ul of this mixture was then injected Zorbax ODS column, with effluent monitored at 220nm. onto the DuPont Angiotensin I elutes at 4.4 min, while the tetradecapeptide substrate elutes at 6.8 min in the above solvent system. Activation of Secreted Recombinant Human Prorenin--Serum free culture media from 293 cells (3.6 1) was treated with EDTA (final concentration 5 mM) and N-ethylmaleimide (final concentration 0.5 mM) with stirring at 4OC for one hour. The cell culture media was then concentrated to 270 ml by tangential flow ultrafiltration (Pharmacia/Filtron) using an Omega 8~ membrane. TPCK-trypsin (0.75 mg/ml) was added to the media, and activation of the secreted prorenin to renin was allowed to proceed for one hour at 40~ with continuous stirring, at which time the trypsin was inactivated by addition of PMSF to a final concentration of 1mM. The solution containing activated renin was filtered through a Nalgene 0.2 u sterile filter apparatus. Purification of Recombinant Human Renin--Purification of reco&inant human renin using affinity chromatography was similar to the method of McIntyre & A. (21) except for the changes described below. The renin (H-113) (22) was used as the inhibitor His-Pro-Phe-His-Leu w [CHZ-NH]Val-Ile-His affinity matrix instead of H-77 due to its specificity for human renin, and was covalently coupled to Sepharose as described by McIntyre et al. (21). The 376

BIOCHEMICAL

Vol. 171, No. 1, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

activated renin solution was loaded at 0.2 ml/min onto an affinity column (5 x 1.2 cm) previously equilibrated in 0.05 M Tris*HCl buffer, pH 7.4. Fractions (5 ml) were collected, and column effluent was monitored at 280 nm, 0.2 AUFS. The column was washed successively with 75 ml 0.05 E Tris.HCl (pH 7.4), 75 ml of 0.05 B Tris-HCl (pH 7.4)/l a NaCl, and then 75 ml of 0.1 B NaOAc (pH 6.0). In all cases, the flow rate was maintained at 0.2 ml/min and 5 ml fractions were collected. Bound human renin was eluted from the H-113-Sepharose column by applying a decreasing pH gradient of 0.1 B NaOAc from pH 6.0 to 4.3 (65 ml of each buffer), followed by a wash with 100 ml of the pH 4.3 buffer. Fractions containing renin activity (as determined by HPLC) were pooled, adjusted to pH 6.5 with Tris base, and the pool was then concentrated to a volume of 8 ml using an Amicon stirred ultrafiltration cell and a YMlO membrane. Kinetic Data--The enzyme turnover number (kcat ) and Michaelis constant for tetradecapeptide substrate (I&) were measured in 0.1 B sodium acetate buffer pH 6.0 at 37OC. Assays were performed with 25-125 u@ tetradecapeptide substrate in 0.05 ml buffer. Renin (0.02 ug) was added to the reaction mixture, and the reaction was allowed to proceed until 30-60% of the substrate at which time a 20 ul aliqout was withdrawn, added to 40 ul HPLC was used, running solvent (above), and 50 ul of this mixture was then analyzed by HPLC as described above. Controls showed that the levels of angiotensin II produced in the reaction did not inhibit the enzyme. RESULTS AND DISCUSSION Although of

enzyme

isolated

recombinant carry

human renin is

detailed

previously

been

human

Shown in recombinant

Figure

human

enhancer

transformed

in

paper, cell

As

an

human

the

transfected expression

in

1 is

phRN-8,

a vector

The expression was

recombinant

cell

lines

several

individual

clones

were

cytodot

hybridization

resistant

to

For

comparative

Eco

RI/,BamHI

HI ,’

purposes,

1.

Vector

for

regions are indicated and mRNA transcripts

the

expression

by open boxes, by arrows.

of

the

377

for

coding

adenovirustransfection,

the the

isolated, renin

and cDNA by

nontransfected

RI

HI

human

sequences

from

ElA-responsive

n-mr.

cDNA

cells

cDNA for

BCII

protein

has

renin

of the

were from

to

renin

human

the

hygromycin

expression

utilized

(CHO)

ElA-producing

Barn

--Figure

of

utilizing

Barn HIlEco

Barn HIlStui

ovary

phosphate-mediated

for

have

level

sufficient

expression

the

analyzed

analysis.

the

into

293 by calcium

stable

Nde I/Barn

for

the

human

hamster

vector,

introduced

line

we quantities

and purification

described.

renin.

(3,4),

alternative, renin

Chinese

293 is

kidney

human kidney

Recombinant

line

(25),

human

from

characterization.

expressed

kidney

low.

to express

protein

In this

(23,24).

BRVPZ

very

DNA technology

out

the

can be purified

renin.

Regulatory

by closed

boxes,

Vol.

171, No. 1, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

B 0.8 H -TPCK

Trypsin

0 + TPCK Trypsin

r1

Expression Figure 2. (A) and secretion of

total

cellular

renin probe.

specific (B)

hygr

stable

and

after

parental

RNA from transformants

line

also

mFWA could

be detected

in the

parental

not

express

the

activity, with

dependent addition, before

(RN) and nontransfected

in

the

if

renin the

of renin

As shown

Figure

conditioned

culture

gene

renin with

resistant Thus,

293

renin of

293 cells cells

for

32P-labelled and four

activity

the

before

lines

was

from

were

Hygr

determined

by

cell

renin

clones

does

assay.

secreting were and

but

line

by this

tetradecapeptide

activity in TPCK-trypsin 378

nine

specific

transformants

human kidney

293

cell

2A,

cell

detectable

medium I

in

at a level

synthetic

angiotensin

the presence of and after treatment

line.

recombinant

angiotensin-containing of

the presence

hygromycin

293 cell

endogenous

release

for

human Analysis

TPCK-trypsin.

was analyzed.

cell

determine the

an

transfected (RN l-4)

with

not

To

of mRNA for recombinant (A) human kidney cells.

in

mFNA by a dotblot procedure using a renin-specific Analysis of conditioned culture media from 293

treatment

293

and identification prorenin (B)

renin incubated

the

renin-

HPLC analysis.

the conditioned was determined.

culture As

In medium shown

in

Vol.

171, No. 1. 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

50

80

-

0.2-

II I 0.15-

! 0 O.l2 a

0.05-

0.

20

10

30

40

60

70

I-

l( lo

90

Elution profile of recombinant human renin from an H-113-Sepharose c01uml. Activated renin was applied to the affinity column and sequentially with 0.1 M Tris HCl (pH 7.4) (arrow I), 0.1 M Tris HCl (pH 7.4)/l M NaCl (arrow II), and 0.1 M sodium acetate (pH 6.0)/l M NaCl (arrow III). Henin was eluted with a pH gradient from pH 6.4 to pH 4.3, followed by an isocratic wash with 0.1 M sodium acetate pH 4.3 (arrow IV). Effluent was monitored at 280 nm for eluted protein (O-O-W). Activity ( W) was determined using HPLC by measuring the area Eunder the peak corresponding to angiotensin I as indicated under "Materials and Methods".

Figure affinitJl washed

Figure

3.

2B,

no

recombinant from

the

that

secreted

the

and

hygromycin

gradient

on

apparent

homogeneity 9.68

(Table

activated below.

from the

in

1.32

The

protein,

The

final

observed

for

CHO-derived

(23). produce

One 100

unit nmol

the

mg

months.

of

31,

stored

angiotensin

at

activity activity I/hour

the

at

379

pH

4OC,

6.5

to purify of

in

the

was

is

using the

amount

and

31°C

for

a yield

stable

for

comparable

a similar of as

renin measured

to

activity

unitslmg to

pH

renin

specific

1495

units/mg

cells.

of a decreasing

purification

renin as

ug/106

was utilized

to

at

1495

defined

(12).

0.2 to 2.0

an increase

6.5

human is

the

from

corresponding

of

recombinant

by

secreted

filtrate pH

phRN-8

confirmed

rapid

was obtained,

with

data

was

from

in

concentrated

renin

specific renin

resulting

and medium

These

transfected

This

for

parental in the

trypsin.

The application

allowed

the

of prorenin

method

medium.

column

(Figure

units/mg

I) %

culture

with

renin. ranged

from

be detected

stably

The level

chromatography

cell

affinity

renin;

lines

transformants

the

could

treatment

293 cell

not

in medium

activity

following

described affinity

renin

purified

lines

resistant

A single-step

be detected

However,

recombinant

analyses

active

from

lines. cell

prorenin

bi0chemica.L

could

activity

recombinant

suggested

nine

renin

293 cell

the

of

16%

several

to

that

HPLC assay needed by

to HPLC

Vol.

171, No. 1, 1990 Table

I.

BIOCHEMICAL

AND BIOPHYSICAL

Purification

of

Recombinant

units Activated Filtrate Final

Prior

to

activated

to

renin

trypsin

resulted

the

were

observed

nicked

in to

with

about

with an

internal

sire

vitro

under

reducing

Da and

in vitro

32,500 trypsin

10 1495

step,

the

activation

of

cleavage

purified of

100

154

76

secreted

prorenin

gel

electrophoresis

that suggesting

prorenin

the

41,000 (3)

to

renin as 4).

corresponded

renin

a

using

However, renin

Under

single is

to molecular

the

was

chain.

Da which

(Fig. that

vield 1

migrated

about

renin

conditions Da,

of

renin

weight

of human kidney-derived

42,000

by the

the

a molecular

fold

SDS-polyacrylamide

trypsin. b

Renin

U/am

1.32

chromatography

that

conditions,

corresponding

of

affinity

Human

269

1973

revealed

4)

2613

Renin

the

(Figure nonreducing

(cone)

mq

RESEARCH COMMUNICATIONS

chain

band

consistent two

bands

weights had

activation.

94 67 43

Figure 4. SDS/polyacrylamide gel electrophoresis of purified recombinant human renin. Lanes 1 and 4, standard M, marker proteins (Pharmacia LMW Calibration Kit): 80 ng each of phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), and alpha-lactalbumin (14.4 kDa); lane 2, 130 ng renin in the presence of 0.5% (wt:vol) dithiothreitol; lane 3, 130 ng renin.

380

been

Vol.

171, No. 1, 1990

BIOCHEMICAL

Table

II.

AminOterminal Sequence Sequencing was performed

Primary Cycle 1 2 3 4 5 6 I 8 9 10 11 12 13 14 15 16 17 18

Amino

Secondary

Yield(pmo1) 360.9 42.4 198.1 72.0

Acid Leu TY~ Thr Ala (CYS) Val TY~ His LYS Leu Phe ASP Ala Ser Asp Ser Ser Ser

C

= 187.8 = 91.9%d

pm01

aThe primary sequence corresponds secondary sequence corresponds to for

Leu

in

cycle

Yield(pmo1) b 112.6 29.1 69.9 C

129.8 141.1 42.5 148.4 176.3 132.2 133.2 54.8 16.5 43.4 29.9 15.1 8.3

(93.9%)

to residues residues 54-71

accounts

for

l-18 of of human

both

human renin.

renin

repetitive 2,7,15),

Leu

Aminoterminal beginning

with

chains

are

Asparagine5

in

a manner

and Cys58

is to

single-chain enzyme

of

the

binding affect human

as a result outer

site. enzyme renin

activity. does

not

of the cleavage

Aside require

activation.

381

from further

X-ray

was fully

Cys58. with

active

(as

by pepstatin

structure bond

protein

and

in

region

removal

and

three-dimensional

disulfide this

is the

consistent

A

renin.

the

of the

surface

Therefore,

renin

renin and that

and was inhibited upon

approximately

Cys51 is

Tyr

sequences

purified

between which

human

based

two

in

the

in is

for 6,lO).

and Leu54,

bond

Two chain

single-chain

formed

that

sequencing,

of

peptides

Arg53

a disulfide

renin,

on the

confirm

tetradecapeptide)

to

presence

both

between

(23,26).

the

human

loop

located

substrate

expected

of

data

the

position

analogous

that

in

the

with

located by

seen

not

model

These

site

together

by cleavage

suggests the

held at this

structural

II).

cleavage

Was

glycosylation measured

(Table

the

showed

respectively,

the

was expected that Asn5

as the mean of the yields (cycles 4,13) and Val (cycles

analysis

and Leu54,

amounts

nicked, two

sequence

at Asp1

equimolar

yield was determined (cycles 3,12), Ala

and

peptides.

CAsn was expected in cycle 5 for the primary peptide while Cys cycle 5 for the secondary peptide. A blank cycle suggests glycosylated and that Cys58 is involved in a disulfide linkage. dAverage (cycles

Renin

Sequenced

Amino

35.8 72.6 16.7 11.7 109.2 67.4 122.0 30.3 28.2 54.2 71.0 42.5 14.5

yield yield

RESEARCH COMMUNICATIONS

of Recombinant Human on 200 pm01 protein

Sequence=

Acid Leu Thr LeU GUY (Am) Thr Thr Ser Ser Val Ile Leu Thr A5n TY~ Met ASP Thr

Theoretical initial Average repetitive

bTh e yield

AND BIOPHYSICAL

of

proteolytic

of

pepsin,

between is

removed might

the

Cys51 from not

be

propeptide, cleavage

for

Vol.

Kinetic synthetic 1.57)

analysis

x 103/hr

against

in

with

0.1

M sodium

a Km of 11.7

is

inhibitor

could

ability

to

be

express

allow

for

for

enzymatic

the

in

the

Thus,

rapid

production

of therapeutically

useful

kcat

These

hypertension

= (7.48

findings

for

5 are

human renin

because

degradation the

mg/l

for

(27).

to

levels

of large

catalyzes and because

a specific

renin

hypertension.

a human

quantities

crystallographic

of

control in

it

cascade

development

useful

characterization,

u&j while

pH 6.0.

angiotensinogen

at

human renin

by radioimmunoassay

to combat

renin

recombinant

buffer,

therapeutically

RESEARCH COMMUNICATIONS

+ 9.3

reported

enzyme.

human

57.1

acetate

target

specific

Km for

was

previously

step

a highly

the

uM determined

an attractive

rate-limiting is

AND BIOPHYSICAL

that

substrate

tetradecapeptide Renin

the

determined

tetradecapeptide

consistant

it

BIOCHEMICAL

171, No. 1, 1990

of

kidney

purified

studies,

and

The cell

will

human

renin

the

development

inhibitors. ACKNOWLEDGMENTS

The authors wish Faust for providing us Fariborz Mohamadi for for growing the 293 sequence analysis.

to express their gratitude to John Chirgwin and Phyllis with the human preprorenin cDNA, to Paul Gesellchen and the synthesis of H-113, to George Boder and Jim Putnam cells, and to Mark Slisz for the gas-phase protein

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