Ge,ir. 104 (1991) 271-275 ic‘ 1991 Elsevier Science Publishers

GENE

B.V. All rights reserved.

271

0378-I 119/91/$03.50

05068

Production of functional rat HMGl protein in Escherichia (High-mobility

group-l

protein;

recombinant

DNA;

protein/DNA

coli

interactions)

Marco E. Bianchi Dipartimento di Genetica e Microbiologia, Universititdi Pavia. I-27100 Pavia (Italy) Received by J.-P. Lecocq: 28 January 1991 Revised/Accepted: 4 April/l5 April 1991 Received at publishers: 23 May 1991

SUMMARY

High-mobility group-l protein (HMG 1) was produced in Escherichiu colt’ under the control of the T7 promoter / T7 RNA polymerase system. The protein can be produced and purified with yields similar to those obtained from animal tissues. HMGI purified from E. coli is homogeneous and capable of selectively binding cruciform DNA, indicating that posttr~slationa~ processing of vertebrate HMGl is not necessary for its DNA-binding ability.

INTRODUCTION

High-mobility group-l protein (HMGI) is a nuclear protein which is extremely conserved in all vertebrates, but whose function has not been identified unequivocally (see Einck and Bustin, 1985, for a review). We recently found that HMG 1 selectively binds to cruciform DNA, a specific conformation of DNA that arises as an intermediate in genetic recombination or as a consequence of torsional stress on paIindromic sequences (Bianchi, 1988; Bianchi et al., 1989). Although HMGi is fairly easy to obtain by extraction of animal tissues with strong acids, these prepa-

Carres~nnderzce ia: Dr. M.E. Bianchi, biologia, Universitl

Dipartimento

di Pavia, via Abbiategrasso

Tel. (+39-382)3915X1; Abbreviations:

bp,

dithiothreitol;

FPLC,

di Genetica

e Micro-

207, I-271 00 Pavia (Italy)

Fax (+ 39-382)528496. base

pair(s);

fast-flow

DEAE,

protein

diethylaminoethyl;

DTT,

liquid chromatography;

4-(2-hydroxyethyl)piperazine-I-ethanesulfomc

acid; HMGI,

Hepes, high mobil-

ity group 1 protein; HMGl, cDNA encoding HMGI; IPTG, isopropyi~-~-thiogalactopyranoside; kb, kilobase or 1000 bp; nt, nucleotide(s); PMSF,

phenylmethylsulfonyl

SDS, sodium

dodecyl

sulfate;

fluoride;

RBS (rbs), ribosome-binding

[ ] designates

plasmid-carrier

state.

site;

rations are not reproducibly active in DNA binding. Even HMGl obtained from rat liver by a gentle, nondenaturing method (Marekov et al., 1984) is heterogeneous with respect to its DNA binding properties, despite migrating as a single band in SDS-polyacrylamide gels. A subpopulation of HMGl molecules can bind to cruciform DNA, while a large fraction of the preparation is uncapable of DNA binding (M.E.B., unpublished results). The protein fraction which binds to DNA can be reversibly denatured and renatured, whereas denaturation/renaturation cycles do not restore DNA binding ability to the inactive protein fraction. This behaviour can be attributed either to sequence microheterogeneities of the HMG 1 molecules (perhaps encoded by different genes), or to post-translational modifications. At present, no evidence exists for the former hypothesis; on the other hand, acetylation, glycosylation and ADP-ribosylation of HMGl have been reported (Reeves et al., 198 1). To prove that the unmodified polypeptide is indeed capable of binding to cruciform DNA, and to secure a source of chemically homogeneous protein, we expressed an HMGl cDNA clone in bacterial cells. The protein purified from the overproducing strain does bind selectively to cruciform DNA.

272 EXPERIMENTAL

AND

DISCUSSION

Production of rat HMGl in Eschevichia coli Initial attempts to produce HMGl in E. c,oli under the control of the luc of tv/) promoters/operators of various expression vectors were unsuccessful. To obtain a tighter

(a)

control of HMGI expression, we then decided to exploit the specificity of T7 RNA polymerase for T7 promoters. The expression vector we chose, pT7-7. is a derivative of pT7- 1 (Tabor and Richardson, 1985). It contains a fragment of T7 gene 10 comprising the T7 RNA polymerase promoter, the RBS, and the ATG start codon followed by a polylinker. We cloned the rat HA4GI cDNA from pRNHMG I (Bianchi et al., 1989) between the NdeI and HijldIII sites of pT7-7, so that the start codon of HMGl replaced cxactlq that of T7 gene 10. The resulting plasmid, pT7-RNHMG 1 (Fig. I), was introduced into E. coli AR68[ pGP I-21, which

pT7-RNHMGl

bla

\

ColEl

on

TTCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTTAG rbs

Met Gly Arg

AATAATTTTGTTTAACTTTAAGAAGGAGATATACAT

atg ggc aaa

agtgtc

tttttttgtatagttGGGGATCCTCTAGAGTCGAGCGGCATGCAAGCTTATCATCGAT

Fig. 1. Map RNHMGI

and

sequence

was obtained

of plasmid

pT7-RNHMGl.

by introducing (HMGI

cDNA coding for HMGl

). The shaded

box represents

region ofHMGI, the open box the 3’-untranslated is the gene that confers resistance to ampicillin origin of replication (T7 promoter.

of the plasmid

arrowhead)

Plasmid

pT7-

into vector pT7-7 a part of the

(ColEl

ori, black dot). the promoter

and the RBS (r/x, blackened

of phage T7, and the start codon of HMGI

the coding

region. Also Indicated (hkr. thick arrow), the

in upper-case

(Paonessa

et al., 1987; accession

is indicated

in lower-case

letters.

The sequence

from rat HMGI

cDNA

No. YO0463 in the EMBL Data Library)

letters and extends

for 792 bp from the trans-

lation start site to 0.15 kb into the 3’.nontranslated region. The sequence in upper-case italics is a piece of the plJC19 polylinker and has been carried

over into pT7-RNHMGI

entire 924.bp sequence

as a result of the cloning strategy.

has been deposited

No. M63853 (rat high-mobility cds).

group-l

in GenBank

protein

synthetic

Similar problems are often encountered for the production of proteins which are toxic to E. cd, since the basal level of T7 RNA polymerase activity in uninduced cells can provide sufficient transcription of the target gene. The E. cdi strain BL21(DE3) harboring plasmid pLysE was specifically developed to solve these problems (Studier and Moffatt, 1986). In this strain T7 RNA polymerase is under the control of the inducible bcUV5 promoter, and its basal level of activity is specifically inhibited by T7 lysozyme, encoded on plasmid pLysE. Upon induction with IPTG, newly made T7 RNA polymerase titrates out its inhibitor, and then drives the expression of the target gene. We introduced pT7-RNHMGI into BL21(DE3)[pLysE]; cells were grown with vigorous agitation in LB medium containing chloramphenicol and ampicillin at 37°C. After reaching an absorbance of 0.7 (at 600 nm), the culture was induced with 0.5 mM IPTG and grown for an additional 2.5 h. Crude cell lysatcs were fractionated by SDS-polyacrylamide gel electrophoresis and either stained directly or analysed by Western blotting with affinity-purified polyclonal anti-HMG 1 antibodies (Fig. 2). Induced BL21(DE3)[pLysE; pT7-RNHMGl] cells contained several immunoreactive polypeptides, absent from control samples. The band of highest M, was indistinguishable immunologically and elcctrophoretically from HMGl prepared from rat liver. The same band was visible in Coomassie stains of the SDS-polyacrylamidc gels, representing about 0.2 to I U, of the total protein content. However, the bulk of the immunoreactive material formed a diffuse band of higher mobility, which presumably derived from proteolysis of HMG I. Time courses of HMG I synthesis after induction confirmed a precursor-product relationship between the polypeptides of higher and lower M, (not shown), and indicated that the best yield of intact HMGl is obtained about 135 min after induction.

box) of gene IO

(ATG. thick margin of the box

representing HMGI ). The lower part of the figure shows the junctions between the vector and the insert. The sequence deriving from pT7-7 is indicated

contains the T7 RNA polymerase gene under the control of the /z pI, promoter and the temperature-sensitive Cl857 j. repressor (Tabor and Richardson, 1985). Although pT7RNHMGI was maintained in the AR68[pGPl-21 cells and after induction the T7 RNA polymerase was synthesized at high levels. we obtained low and irreproducible synthesis of HMG 1 protein.

The

under accession gene, complete

Purification of rat HMGl produced in Eschevichia coli A culture of BL21(DE3)[pLysE; pT7-RNHMGI] (10 liter) was grown in a pilot fermentor. Growth and induction were essentially identical to the small-scale conditions described in the preceding paragraph. The packed cells (45.7 g) were resuspended in 225 ml of buffer D-400 (20 mM Hepes pH 7.9/0.4 M NaCI:‘0.2 mM EDTA/0.2”,, nonidet-P40/l mM DTTjO.5 mM PMSF’IO”,, glycerol), frozen in liquid nitrogen, thawed and vigorously stirred. After an additional freeze/thaw cycle, 12 g of DEAE-cellu(b)

213 ammonium 97.4

sulfate/20

mM

Hepes

pH

7.9/0.2 mM

. 42.7

EDTA/O.S mM DTT. The material reactive against antiHMGl antibodies eluted at about 0.6 M ammonium sulfate. The pooled fractions (fraction II. 2 ml) were reduced to 0.3 ml with a Centriconmicroconcentrator and applied to a 30-ml Ultrogel AcA54 column equilibrated with 20 mM Hepes pH 7.9/200 mM KCl/0.2 mM glycerol. The fractions conEDTA/O.S mM DDTj5”;

- 31 .o

taining HMGl were pooled and labeled fraction III. Fraction III contained about 200 pg of HMG 1 protein, appeared to be more than 9504, pure (Fig. 3) and comigrated in SDS-polyacrylamide gels with HMGl purified

97.4

66.2 . 66.2 42.7

‘31 .o

from rat liver (data not shown). 21.5

- 21.5 . 14.4

- 14.4 Fig. 2. Production 37°C

and BLZl(DE3)[pLysE]

in LB broth

absorbance

in E. coli cells. Strains

of HMGl

pT7-RNHGMI]

in the presence

of antibiotics.

of 0.7 at 600 nm, both cultures

for 2.5 h in the presence

or absence

cells were then collected

and dissolved

loaded

were stained

rabbit

affmity-purified

goat anti-rabbit phatase.

L.anes:

l-5,

BL21(DE3)[pLysE] 2, induced

polyclonal

antibodies

duced

BLZl(DE3)[pLysE;

mentioned

points

antibodies,

proteins.

most

cells.

HMGl

prominent

in section b. Size markers

6 and

7, proteins cells; 7, in-

cells. The blackened

produced

- 42.7

from rat liver;

cells; 5, uninduced

Lanes:

proteolytic

- 66.2

phos-

1, uninduced

BL21(DE3)[pLysE]

pT7-RNHMGl]

- 97.4

BLZl(DE3)[pLysE]

pT7-RNHMGl]

blue; 6, induced

to full-length

to the

HMGl

to with

biotinylated alkaline

Lanes:

cells; 3, uninduced

pT7-RNHMGI]

with Coomassie

arrowhead

blue or transferred

were immunostained

anti-HMGl

immunostained

stained rowhead

proteins

BLZl(DE3)[pLysE;

1234

100 pg of total protein, were gels. After electrophoresis,

cells plus 50 ng of purified

BLZl(DE3)[pLysE;

grown

(0.5 mM IPTG). The

and streptavidin-conjugated

BLZl(DE3)[pLysE]

cells; 4, induced

an

were split and further

directly with Coomassie

filter. The transferred

at

attaining

directly in loading buffer. Samples

cells, containing about polyacrylamide on O.l?o SDS-15%

an lmmobilon

were grown

Upon

of the inducer

from the dissolved the proteins

BL21(DE3)[pLysE;

(as a control)

(c) DNA binding activity of rat HMGl produced in Escherichia coli The DNA binding properties of the material present in fraction III were compared to those of HMGl obtained from rat liver by the procedure of Marekov et al. (1984) and further purified by affinity chromatography on cruciform DNA-Sepharose (for details about this step of purification, see Bianchi et al., 1989). Both protein samples bound cruciform DNA (molecule C; Bianchi, 1988) with similar efliciency, but did not bind linear DNA (molecule B), or single-

-31.0

ar-

in E. coli, the open degradation

on the right margins

product, are in kDa.

- 21.5 lose equilibrated with buffer D-400 were added to the viscous lysate and stirred. This slurry was centrifuged for 3 h at 4°C in a Ti45 rotor at 45000 rpm. The supernatant (200 ml) was diluted with an equal volume of water and mixed with solid ammonium sulfate to a final concentration of 2.8 M. The resulting suspension was centrifuged at 4°C for 15 min in a Sorvall GSA rotor at 15 000 rpm. The supernatant (fraction I, containing about 50% of the intact HMGl originally present in the cell lysate) was filtered, applied to a Phenyl-Superose HR5/5 FPLC column and eluted with a 20-ml linear gradient of 2.8 M to 0 M

- 14.4

Fig. 3. Purification various

stages

of HMGI

of purification

produced were loaded

by E. co/i. Samples

acrylamide gel, electrophoresed, and silver-stained. BLZl(DE3)[pLysE; pT7-RNHMGl] cells dissolved buffer;

2, the supernatant

after ammonium

from the

on a 0.19,, SDS-15%

sulfate

Lanes: directly

poly-

1, induced in loading

precipitation

(frac-

tion 1); 3, the pool after fractionation on Phenyl-Superose (fraction II); 4, HMGI after fractionation on Ultrogel AcA54 (fraction III). Size markers

on the right margin

are in kDa.

274 stranded oligodeoxyribonucleotides (Fig. 4 and results not shown). Since all protein molecules freshly obtained after an affinity puri~cation step must be able to bind to their ligand, the result displayed in Fig. 4 implies that nearly loo”,, of the protein produced in E. coli and purified described in section h is active in DNA binding. (d) Conclusions Mammalian HMG 1 protein has been produced

as

in E. co/i

cells. This result could be obtained only by using the expression system that can be regulated most tightly, which is based on the absolute specificity of T7 RNA polymerase towards activity

its promoters and on the inhibition of its basal by T7 lysozyme. It is possible that even low

amounts of HMG 1 could be toxic to E. colicells by binding to its DNA.

I

Fig.4,

DN,4

I

I+l+l+

binding

activity

contained

mixtures

10 mM

linear DNA

of HMGI Hepes

produced

7.9:8”,, ~icoll~l5~ mM spermidineiO.5 mM mM

MgCQl nonidet-P40

EDTAIl mM and son&ted salmon

nonspecific

competitor.

“P-labeled

mM

by E. co/i. Assay

pH

DTT:O.O5”,,

NaCl/lO

1

DNA

cruciform

DNA

( 100 &ml) (molecule

as C;

Bianchi. 198X)or linear DNA (molecule B)wcrc added to a final conccntration of 0.1 !iM. HMG I protein purified from E. coli (fraction III) or from rat liver (including sepharosc)

an unity-puri~catioll

step on cruciform

wcrc mixed on ice to the assay mixtures

concentration assay mixtures

of 5 pgjml. After incubation acid/EDTA

on ice for 5 min, 6 ill of the

buffer. Electrophorcsis

HMGI-DNA

bands with lower mobility.

complexes.

I am grateful to Monica Beltrame and Vincenzo Notarianni, who participated in some preliminary parts of this work. I would like to thank Dr. Michael Stark and his collaborators at the University of Dundee (U.K.) for making available their pilot fcrmentor and their help. This work was supported by funds from Minister0 dell’Universit8 c della Ricerca Scientifica e Tecnologica of Italy. I was a recipient ofthe EMBO Short Term Fellowship ASTF 6410 while in Dundee.

DNA-

was performed

gel

when they are present,

REFERENCES

at

room temperature for 3 h at I? V/cm. The gel was then fixed in IO”,, acetic acid, dried and autoradiographed with Kodak XAR-S fitm at +O”C with intcnsifyi~~g screens. The lowermost band in each lane is free DNA: the fainter

suggest that the unmodified polypeptide chain of HMGl can bind to DNA and that the fraction of animal HMGl incapable of DNA binding is most probably covalently modified. An alternative explanation is that the available cDNA clone for rat HMG 1 codes for an alternative species of HMG 1, while a large part of cellular HMG 1 would be encoded by a different gene or allele. This interpretation is extremely unlikely since the sequence of the existing rat HMGI cDNA clone is completely homologous to HMGI cDNA clones from human and beef (Wen et al., 1989; Kaplan and Duncan, 1988). The purification procedure we report here is simple and fast, and requires materials which are all commercially available. We also applied it with success to the production of HMGl labeled with [ ““Slmethionine, which cannot be obtained easily from animal sources. We believe that the availability of a convenient source of ho~logeneous HMGl will be extremely valuable in the study of the physiological role of this protein, which is at present not clear.

to a final estimated

were loaded onto a 1 mm thick, 6.5”,, polyacrylamide

in 0.5 x Trisiboric

The yields obtainable from our HMGl expression strain are not extremely high, most probably due to the rapid degradation of the newly synthesized protein within the cells. However, such degradation problems are also encountered during the purification of HMG 1 from animal sources. The HMGI obtained from E. coli is not expected to contain any post-translational modi~cation, and is thercfore molecularly homogeneous. Its ~hromato~raphi~, electrophoretic and DNA binding properties are indistinguishable from those of HMG 1 purified from rat liver by means of affmity chromatography on cruciform DNA. These data

are

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