Gene expression, purification and characterization of recombinant human neutrophil collagenase

Gene, 146(1994)297-301 0 1994 Elsevier Science B.V. All rights reserved.

297

0378-l 119/94/$07.00

GENE 08072

Gene expression, purification and characterization neutrophil collagenase

of recombinant human

MMP-8; recombinant DNA; prokaryotic expression; inclusion bodies)

(Metalloprotease;

Thau F. Hoa, M. Walid Qoronfleha, Robert C. Wahlb, Tricia A. Pulvinob, Karen J. Vavra”, Joe Falvoc, Tracey M. Banksa, Patricia G. Brakea and Richard B. Ciccarelli” Departments ofaMolecular Biology, and bEnzymology, Sterling Winthrop Pharmaceuticals Research Division, Collegeville, PA 19426-0900, USA. Tel. (I -610) 983-7146; and ‘Eastman Kodak Company, Rochester, NY 14650-2158, USA. Tel. ( l-71 6) 588-0735 Received by G.P. Livi: 21 September

1993; Revised/Accepted:

22 March/l

April 1994; Received at publishers:

5 May 1994

SUMMARY

Human neutrophil collagenase (HNC) is a member of a family of matrix metalloproteinases (MMP). HNC is capable of cleaving all three a-chains of types I, II and III collagens. In rheumatoid and osteo-arthritis, MMP members have been implicated in the pathology associated with these diseases due to the accelerated breakdown of the extracellular matrix of articular cartilage. A cDNA coding for the HNC catalytic domain (lacking both the propeptide and C-terminal fragments) was sub-cloned into the pETlla prokaryotic expression vector. The cloned fragment encodes a protein that extends from amino acids (aa) Met”’ through Gly 262of the full-length proenzyme, which as a result, would not require proteolytic or chemical activation. The HNC construct was expressed in Escherichia coli and recombinant mature, truncated neutrophil collagenase (re-mNC-t) was produced at high levels (approx. 30% of total bacterial protein). The re-mNC-t protein was extracted from inclusion bodies by solubilization in 6 M urea, followed by ion-exchange chromatography. The protein was refolded to an active conformation in the presence of Ca2+ and Zn2+. A final purification step on size-exclusion chromatography yielded 30 mg per liter of active re-mNC-t with minor autodegradative products. Alternatively, hydroxamate affinity chromatography was used to obtain pure, non-degraded re-mNC-t (20-25 mg per liter). The catalytic activity of re-mNC-t was abolished by known MMP inhibitors and the Ki measurement against actinonin was similar to that of HNC prepared from human blood.

INTRODUCTION

Enzymes in the matrix metalloproteinase (MMP) family are secreted as zymogens, are inhibited by tissue inhibitor of metalloproteinases (TIMP), require Zn2+ for Correspondence

to: Dr. M.W.

Qoronfleh,

Department

of Molecular

Biology, Sterling Winthrop Pharmaceuticals Research Division, P.O. Box 5000, Collegeville, PA 19426-0900, USA. Tel. (l-610) 983-5620; Fax (I-610) 983-5293. Abbreviations: A, absorbance (1 cm); aa, amino acid(s); BCIP, 5-bromo-4-chloro-3-indolyl phosphate; bp, base glutathione S-transferase; HFC, human fibroblast HNC, human NC; Ig, immunoglobulin; IPTG, thiogalactopyranoside; kb, kilobase or 1000 bp; LB, SSDI 0378-l

119( 94)00283-X

Ap, ampicillin; pair(s); GST, collagenase; isopropyl-fl-oLuria-Bertani

catalysis and need Ca 2c for stability. In addition to their high primary sequence homology, they are structurally conserved and most are arranged into three functional domains. These domains consist of a propeptide region with a cysteine residue responsible for maintaining lat(medium);

MMP, matrix metalloproteinase;

M9, minimal

salts medium;

NBT, nitro blue tetrazolium chloride; NC, neutrophil collagenase; NC-t, NC C-terminal-truncated; oligo, oligodeoxyribonucleotide; PAGE, polyacrylamide-gel electrophoresis; PCMB, p-chloromercuribenzoate; PCR, polymerase chain reaction; proHNC, full-length HNC proenzyme (latent); proNC-t, NC-t proenzyme (latent); PUMP, putative matrix proteinase (matrilysin); re, recombinant; re-mNC-t, recombinant mature NC-t; sepragel, SDS-PAGE; SDS, sodium dodecyl sulfate; TIMP, tissue inhibitor of MMP; vvm, volume of air per volume of medium per min.

298 ency, a catalytic

domain

site and a C-terminal pexin (Docherty genase (HNC:

with a conserved

portion

Lint-binding

with resemblance

et al., 1992). Human

to hemo-

neutrophil

colla-

encoding

proteinase

which

glutathione-S-transferase

EC 3.4.24.34) is a neutral

belongs to the MMP enzyme family. The MMP members are capable varying

of degrading

specificities

et al., 1993). The MMPs role

native

collagen

remodelling.

stems from their ability

The

to destroy

This

interest

matrix.

destruction

diverse pathological

occurs

states such as inflammation,

tis, metastasis

and peridiontal

disease (Henderson

1990). Several

pharmaceutical

companies

a number

of the MMPs

for diseases principal

for the development

where loss of connective-tissue

and generates (GST)

arthriet al.,

have targeted

with the desired 1988) domain

authentic lacks

genase (MMP-1)

eliminated.

HNC with these proteins

neutrophil is activated to exocytosis (Hasty et al., 1990; Sorsa et al., 1992). The synovial fluid in rheumatoid and osteo-arthritis is characterized by the presence of abundant neutrophils. The local release of neutrophil type MM P-8 collagenase and its effect on type II collagen found in articular cartilage is thought to contribute to the progressive destruction of joint tissues (Sorsa et al.,

Stromelysin

Therefore, strategy

we due

to

(MMP-3) colla-

adopted

the

homology

of

and chose the T7 polymerase-

directed expression system (pET1 la vector. Studier ct al.. 1990) to generate authentic HNC protein. DNA encoding C-terminally truncated forms of HNC (proNC-t and mNC-t) were PCR-amplified from pGEX-2T/hnc and sub-cloned into pET1 la (see legend to Fig. 1A). The plasmids were designated pWQlO0 and pWQll0, respectively: the proNC-t-encoding clone clone with begins with Phe”‘, the mNC-t-encoding Met”‘. Both clones end with GlyZh’ (a naturally autolytic site exists between Gly”’ and Leu’“” marking the approx. border between the catalytic domain and hemopexin-like

1992; Henderson et al., 1990). This suggests that HNC is a reasonable target molecule for therapeutic intervention

domain).

in diseases accompanied with accelerated breakdown of the extracellular matrix of articular cartilage. The aim of the present study was the cloning, expression in E. di, purification and characterization of two HNC forms. We succeeded in producing a form of the protein, re-mNC-t, which contains only the catalytic domain, thereby obviating the need for proteolytic or organomercurial compound activation and also removing an autolytic site near the C terminus (for detailed discussion on activation and autocatalysis of MMPs the reader is referred to VanWart and Birkedal-Hansen,

(b) Expression of proNC-t and mNC-t Expression of re-proNC-t and re-mNC-t

1990). The 18-kDa recombinant mature, truncated neutrophil collagenase (re-mNC-t) protein produced is pure, active, and suitable for studies of catalysis and inhibition. In addition, a homogeneous affinity-purified preparation of the truncated protein was used for the X-ray craystallographic studies recently reported by Stams et al. (1994).

et al..

hemopexin-like

(Lowry et al., 1992) are active with this

C-terminally-truncated

secreted

( Muller

MMP-7) C-terminal

and is an active enzyme.

integrity

is a

the

for biophysical

(Ye et al., 1992; Marcy et al., 1991) and fibroblast domain

glycoprotein which is synthesized as a latent proenzyme during the myelocyte stage of neutrophil development. It is stored in the secondary or specific granules until the

EXPERIMENTAL

with

a source of active. re-HNC N terminus

( PUMP;

Matrilysin naturally

of inhibitors

is a 75kDa

protein

fused to the N terminus

(a) PCR cloning of proNC-t and mNC-t forms of HNC

studies. in

feature.

HNC, also referred to as MMP-8,

a fusion

of Ynlc pcptidc-

region ( Phc” ).

of the propeptide

Our aim was to generate in

components

of the extracellular

sequence

Dr. N. Berliner lacks the signal

with

et al., 1991; Hirose

are believed to play a significant

in connective-tissue

MMPs

interstitial

(Netezel-Arnett

pGEX-2T:hnc clone from University. This construct

was carried

out in the E. coli lysogen BL21 (DE3). Cultures were grown at 37 C either in terrific broth for small-scale preparations or in LB + M9 medium for 10 liter fermentations. in the presence of 100 pg Ap per ml, to .4600=0.7p1 .O or 1.3-1.5, respectively, and induced with 0.5 mM IPTG. Aliquots were collected at different time points. The harvested samples were normalized and analyzed on precast IO--20% gradient sepragel. An example of a typical fermentation/expression experiment is presented in Fig. 1A. Routinely, the 18-kDa re-mNC-t accumulated at high levels (approx. 30% of total bacterial protein) after l-3 h induction (Fig. 1A, lanes 2--4). Western blot analysis (Fig. IB, lane 4) using a polyclonal antibody raised against human fibroblast collagenase ( HFC) (BirkedalHansen et al., 1988) showed that re-mNC-t is apparently related to HFC despite the low homology between HNC and HFC (Hasty et al., 1990; Devarajan et al.. 1991 ).

AND DISCUSSION

The cDNA cloning of HNC has been previously scribed (Devara_jan et al., 1991). We obtained

dethe

(c) Purification of re-proNC-t and re-mNC-t Methods for purifying truncated MMPs have been detailed elsewhere (Ye et al., 1992; Schnierer et al.. 1993).

299

c

B kDa

M

1

2

3

kDa

4

M

1

2

3

4

kDa

Ml

234

95.0 94.0

66.0

67.0 43.0

39.0 29.0 20.4

29,o

-

mNC-t

20.4 cmNC-t

mNC-t

14.0 Fig. 1. Production of mNC-t in E coii. (A) Synthesis of re-mNC-t in E. coli. Oligos used to synthesis PCR products coding for pro and mature NC-t included: HNC-1, a 30-mer proenzyme N terminus end, HNC-2, a 27-mer mature enzyme N terminus end and HNC-3, a 36-mer truncated C terminus end. The sense primers incorporated an Ndel site at their S-end for an ATG start codon. The antisense primer possessed a BarnHI site with two TGA stop codons in tandem. HNC-1, S-CTC CAT ATG NdeI Met HNC-2, S-CTC -~ CAT ATG ~deI/M’~ HNC-3, S-CTC GGA TCC BumHI

TTT CCT GTA TCT TCT AAA GAG F2’ TTA ACC CCA GGA AAC CCC TCA TCA TCC ATA GAT GGC CTG AAT GCC __-

stop -+----+----

@?62

The resulting PCR products of a 729-bp fragment (proNC-t) and a 489-bp fragment (mNC-t) were cloned between the N&I and BamHI unique sites of pETlla (Studier et al., 1990; also see section a).The nt sequence of the catalytic domain was verified by double-stranded dideoxy DNA sequencing (Kraft et al., 1988). The growth of NC-t producing E. coli pWQll0 was scaled up to 10 liters in a 15-1Bioengineering AG fermentor. The medium used was LB + M9 and 1 ml Mazu DF204 antifoam agent was added to the medium. The cooled medium was supplemented with a filter sterilized solution consisting of glucose, MgSOd, and Ap to a final con~ntration of 0.4%, 1 mM and 100 &g/ml, respectively. A 200-ml overnight culture was used to inoculate the fermentor. The fermentation conditions were: 37”C, 500 rpm, and an aeration of 1 vvm. The culture was induced with 0.5 mM IPTG when it reached an A,, of 1.3. I-ml samples were taken, pelleted, resuspended in a 100 ~1 2 x SDS sample buffer, boiled for 5 min and centrifuged. A normalizing volume loaded to A of sample was run onto a 10-20% gradient sepragel (IS& Hyde Park, MA, USA) according to manufacturers instruction, then Coomassie blue R-250 stained (see section b). Lane M, ISS molecular mass markers in kDa; lane 1, prior to IPTG induction (0 h); lanes: 2-4 post induction (+ IPTG) at 1.3, 1.7 and 2.3 h, respectively. (B) Western blot analysis of re-mNC-t expressed in E. c&i. Cultures were grown in terrific broth and induced with 0.5 mM IPTG for 2 h. Gels were run onto precast l&20% gradient sepragel then electrotransferred to nitrocellulose paper essentially as described by Towbin et al. (1979). Western immunoblotting was carried out using a rabbit polyclonai antibody raised against HFC (Birkedal-Hansen et al., 1988). Then, the blot was treated with a secondary antibody, an alkaline phosphatase conjugate of goat anti-mouse IgG, and developed with BCIP/NBT (Mierendorf et al., 1987; also see section b). Lane M, ISS molecular mass markers in kDa; lanes 1 and 2, the host strain without the NNC gene (-/+ IPTG); lanes 3 and 4, the host strain with the HNC gene (-/+ IPTG). (C) Affinity purification of re-mNC-t. Methods: inclusion bodies were recovered, solubilized with 6 M urea. and the re-mNC-t was isolated by MonoQ chromatography. The protein was then renatured and concentrated. A Sepharose-Pro-Leu-Giy-NHGH affinity column (Moore and Spilburg, 1986) was used to purify the full length enzyme. The peptide hydroxamic acid moiety is a substrate analogue with a metal-cheIating group allowing the Zn~+-conta~njng enzyme to bind the column (see section c). A 15-mg sample was loaded onto the lo-ml column equilibrated with 20mM Tris/S mM CaCIJ0.4 M NaCI/O.02% NaN, pH 7.5 buffer at a rate of 0.7 miimin. After loading, the column was washed with 3 vols. of the above buffer to remove nonspecifically bound protein. The specifically bound protein was eluted with 4 vols. of 0.2 mM hydroxamate inhibitor dissolved in the same buffer. The eluate was then concentrated in an Amicon stirred cell with a IO-kDa cut off YMIO membrane that also get rid of minor peptide contaminants. This yielded 11mg of pure, active re-mNC-t. Shown in C) is a Coomassie blue R-250 stained lo-20% gradient sepragel. Lane M, Bio-Rad molecular mass markers in kDa; lanes: 1. 5 pg re-mNC-t prior to aflinity purification; 2, 5 pg protein obtained from affinity column flow through; 3, 5 pg protein of pooled elution fractions; 4, 5 pg of re-mNC-t post concentration.

We have adapted these methods for purification of truncated HNCs. Briefly, inclusion bodies were recovered from lysed bacteria (Marcy et al., 1991). The pellet was solubilized in 6 M urea and after centrifugation the supernatant was loaded onto a MonoQ-Sepharose column. Pooled fractions were diluted tenfold and renaturation

was performed with refolding buffer containing Zn*+ and Ca2+ at 4°C. After the protein was concentrated, a final step of purification was carried out on a Sepharcyl S-100 HR column. Minor impu~ties detected on sepragels were attributed to the degradation of the re-protein based on aa sequencing. Whereas, this protocol yielded highly

300 purified

(3 I pg/ml ) suitable

re-mNC-t

studies of catalysis activation

and inhibition

of purified

romercuribenzoate (Mookhtiar

and VanWart,

analysis

of re-mNC-t

mature

HNCs

(approx.

IO-15%

presence

or absence

(see

section

Sepharose following

ligand

permitting products

aa sequence

then

rapid

an abundant

for biophysical

by

known

MMP

nant

nei-

communication),

coupled

with affinity purification

source

of pure, active re-mNC-t

characterization.

(5) This approach

The

resolves many of the difficulties

ciated with low yield (as has been observed

asso-

with recombi-

GST-proHNC;

activation

N. Berliner, personal and the need for enzyme processing and

(Schnierer

et al., 1993).

hydroxamate

and Spilburg, for further

1986) purifica-

coordinates

purification

re-mNC-t and other

is inhibited

(4) E. cdi expression provided

the Met).

activ-

activity

inhibitors.

Met

utilized

(Moore

to

This

initiator

Met influenced

of the re-protein,

to Fig. 1C). Full-length degradation

achieved

(see below) nor enzymatic We

tion. In this step, the metalloenzyme affinity

be

the

lacked

of the initiator

d).

not

or p-chlo-

that it corresponded

without

of the protein

chromatography refolding

could

1990). N-terminal

and

ther affinity purification ity

with trypsin

confirmed

with

enzyme.

(see section d). efficient

re-proNC-t (PCMB)

for mechanistic

to the

was separated contaminants

REFERENCES

(see legend from

(Fig. lC,

lane 3; 1%kDa band), yielding a pure, active, NC-t which could be concentrated without autodegradation (Fig. lC, lane 4). Small-peptide impurities (less than 10 kDa) appearing after affinity purification were removed during concentration. The identity of the protein was confirmed by N-terminal aa sequencing. The affinity-purified enzyme was successfully used for X-ray structure determination (Stams et al., 1994).

Birkedal-Hansen.

substrates as described previously (Stack et al., 1989; Knight et al., 1992). Using the pentapeptide benzoylPLALW-NH-(CH,),-N-(CH,), as a substrate in a fluorimetric assay in a microtiter plate (R.C.W., unpublished data), re-mNC-t had a specific activity of 1.58 + 0.08 pmol product/h/ug protein. The K, and k,,,/K, values obtained are 63.5 f 9.2 uM and 30.4 + 2.2 ).rM ’ h ‘, respectively. The activity of re-mNC-t was inhibited by ion chealators such as 5 mM EDTA and 10 mM I,10 phenanthroline. The inhibitor actinonin (hydroxamate functionality, Wahl et al., 1989) displayed a measured Ki of 160f 10 nM. The native NC isolated from human blood have comparable kinetic (Mallya et al., 1990; NetezelArnett et al., 1991) and Ki values (Wahl et al., 1989). (e) Conclusions (I ) The coding region for proNC-t and mNC-t was amplified by PCR, sub-cloned and expressed in E. co/i utilizing the inducible T7 polymerase PET system. (2) The catalytic domain of HNC (mNC-t) was produced at very high levels in E. coli, purified in a two-step procedure and successfully refolded. (3) The re-mNC-t protein is catalytically active and has kinetic parameters nearly equivalent to the native

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