Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein

21

Gene, 67 (1988) 21-30 Elsevier GEN 02422

Vectors that facilitate the expression and purification of foreign peptides in EscRericRia coli by fusion to maltose-binding protein (Recombinant

DNA;

plasmids;

cross-linked

amylose

affinity chromatography;

starch)

Chu di Guana, Ping Lib*, Paul D. Riggs” and Hiroshi Inouyeb+ “New England BioLabs, Beverly, MA 01915 (U.S.A.) and bDepartment of Biology, Temple University,Philadelphia, PA 19122 (U.S.A.) Tel. (215) 787-8877 Received

31 August

Accepted

31 October

Revised Received

1987

25 February

1987 1988

by publisher

11 March

1988

SUMMARY

Vectors were constructed that allow foreign peptides to be expressed in Escherichia coli as fusion proteins. The peptides are fused to the C terminus of maltose-binding protein (MBP), which allows them to be purified by the MBP’s affinity to cross-linked amylose (starch). The fusion protein can be directed to the periplasm by including the leader sequence from the phoA gene on the vector.

INTRODUCTION

some-binding site and translation start of the 1ac.Z gene. Such hybrid proteins can be purified by affinity chromatography using antibody to /I-galactosidase. An alternative approach to the expression and puriti-

Gene fusions have been used successfully to facilitate the expression and purification of peptides produced from recombinant DNA molecules (Lowenadler et al., 1986; Shuman et al., 1980; Shuman and Silhavy, 1981; Sugita et al., 1985). Most commonly, target peptides have been fused to j&galactosidase, so that the peptide is expressed in Escherichia coli as a fusion protein using the ribo-

cation of foreign peptides is to fuse them to a specific binding protein. The hybrid protein can then be purified by affinity chromatography using the binding protein’s substrate. In the present study, we chose for this purpose MBP, the product of the malE gene of E. coli K-12.

Correspondence to: Dr. P.D. Riggs, New England

phosphates;

Tozer Road,

Beverly,

MA 01915 (U.S.A.)

* Present

address:

Department

Genetics,

Harvard

University

02115 (U.S.A.)

of Microbiology Medical

Biolabs,

32

Tel. (617) 927-5054. School,

and Molecular Boston,

MA

Tel. (617) 732-1921.

maltose

buffer, see MATERIALS

MBP, maltose-binding

Ap, ampicillin; dNTP,

bp, base pair(s); d, deletion;

any of the four deoxynucleotide

Cm,

pyranoside;

tri-

0378-l 119/X8/$03.50 0 1988 El sevier Science Publishers B.V. (Biomedical

Division)

XGal,

liquid chromatography; kb,

kilobases

AND METHODS,

protein; phoA,,

PolIk, Klenow (large) fragment tetracycline;

chloramphenicol;

high-performance

isopropyl-/l-D-galactoside;

gene that codes for the alkaline

t Deceased. Abbreviations:

HPLC,

IPTG,

1000 bp; section c;

, the portion of the phoA

phosphatase of6.

or

signal sequence;

coli DNA polymerase

I; Tc,

5-chloro-4-bromo-3-indolyl$-o-galacto-

[ 1,designates

plasmid-carrier

state; :: , novel joint.

22

Some advantages express and purify

of using fusions to MBP to foreign peptides are outlined

below.

proteins

First, hybrid

single chromatography

can be isolated

step. MBP binds

1982). Second, the materials for aftlnity chromatography are inexpensive and easy to prepare, making large-scale purification simple and feasible. Third, unlike antibody affinity resins, the whole purification takes place under physiological conditions, and protein denaturation upon elution from the affinity matrix should not occur. Fourth, the fused protein can potentially be directed into different cellular compartments by varying the leader sequence of MBP. Finally, MBP does not contain any cysteine residues that could interfere with disulfide bond formation within the target peptide.

AND METHODS

(a) Bacterial strains and culture conditions Bacterial strains are listed in Table I. Bacteria were grown in rich medium containing (per liter) 10 g tryptone, 5 g yeast extract and 5 g NaCl. Antibiotics were purchased from Sigma, and were added when required as follows: 100 pg Ap/ml, 20 pg Cm/ml, 20 pg Tc/ml. The /I-galactosidase indicator, XGal, was added at 20 pg/ml, and the gratuitous inducer IPTG was added to a final concentration of 1 mM

TABLE

and IPTG

were

(b) Expression of the gene fusions and preparation of crude cellular extracts

results in a high yield and a high degree

of purity, as has been demonstrated for MBP itself (Ferenci and Klotz, 1978; Kellerman and Ferenci,

MATERIALS

XGal

in a

with high

affinity to a cross-linked amylose matrix, and is released from the matrix by 10 mM maltose. This purification

to induce the lac promoter. purchased from Sigma.

Cells containing proteins

plasmids

under lac promoter

that coded control

for fusion

were grown to

5 x lo8 cells/ml at 37°C with shaking in rich medium, IPTG was added to a final concentration of 1 mM, and the culture was grown for an additional 2 h. All subsequent steps were carried out at 4’ C or on ice. The cells were harvested by low-speed centrifugation, resuspended in l/l0 vol. of 10 mM Tris - HCl pH 7.2, and lysed by sonication. Cellular debris was then pelleted by high-speed centrifugation, and the supernatant was saved as crude cellular extract. Crude cellular extracts of cells bearing for pCG828 were prepared as described above, except that 1 y0 Triton X- 100 was included in the extraction buffer in case the signal sequence in this construct leads to the association of the hybrid protein with the membrane. For the phoA ss : : malE: :phoA fusion, the strain MZ20 (A phoA A malB phoR [ pPL-6A’ 1) was grown to 5 x log/ml in rich medium and the cells were harvested. A periplasmic fraction was prepared by cold osmotic shock and by spheroplast formation as described by Neu and Heppel (1964; 1965). (c) Cbromatographic teins

purification of the hybrid pro-

Preparation of cross-linked amylose from amylose (Sigma Cat. No. A-9262) and its use as an afI?nity

I

Escherichia coli strains Strain

Genotype

RR1

hsdS20

CG864

RR1 AmalB zjc::Tn5

Source/Reference ara-14proA2

lacy 1 galK2 rpsL20 xyl-5 mtl-1 supE44

Maniatis

lon::TnlOAl6All

this work

SF1362

A(lac)X74

MZ20

A(Iac)X74 phoA20 phoR hpam

araD 139 A(araABC-leu)7679

JM83

ara A(lac-proAB)

71-18

A(lac-proAB)

rpsL (480lacZAM

thi supE [F’

galU galK rpsL thi AmalB zjc::TnS

AmalB rpsL 15)

proA+B+ IacP lacZAM151

et al. (1982)

S. Froshauer J. Beckwith Yanisch-Perron

et al. (1985)

Yanisch-Perron

et al. (1985)

23

chromatography

matrix was performed

as described

by Kellerman

and Ferenci

(1962). Hybrid

were purified

from

extracts

crude

proteins

by binding

to

cross-linked amylose in a 3 cm x 6 cm column, and were eluted with 10 mM Tris - HCl buffer containing 10 mM maltose

(maltose

plasm.

pCG806

sequence,

contains

and potentially

the normal

maZE signal

allows the export

hybrid protein into the periplasm.

High-level

sion of the hybrid protein can be induced IPTG or lactose to the culture medium.

of the expres-

by adding

buffer). (b) Construction of the malE: : 1ac.Z gene fusions

(d) Immunodiffusion A malE : : IacZ gene fusion containing Reagents

for two-dimensional

were purchased was performed

from BioRad, according

and the procedure

to the manufacturer’s

in-

structions. Antiserum directed against MBP was kindly provided by Dr. P. Bassford. Antibody directed against PstI endonuclease was kindly provided by Dr. M. Philipp. Antibody directed against /I-galactosidase was purchased from Promega Biotec.

RESULTS

AND DISCUSSION

(a) Maltose-binding

almost all of

the ZacZ gene (and thus coding for fl-galactosidase

immunodiffusion

protein fusion vectors

To facilitate the construction and expression of protein fusions to MBP, two fusion vectors, pCG150 and pCG806, have been constructed (Fig. 1). Both plasmids are derivatives of pUC18 (Yanisch-Perron et al., 1985) and contain a portion of the maZE gene with (pCG806) or without (pCG150) its signal sequence, fused to the lacZcl coding sequence. The fusion protein is expressed from the lac promoter and confers a Lac + phenotype on strains containing the ZacZdMlS deletion (cr-complementation; e.g., see Yanisch-Perron et al., 1985). The insertion of cloned sequences into the polylinker of pCG150 or pCG806, which is located between malE and lacZa, abolishes the a-complementation. Any peptidecoding sequence can be cloned into the polylinker regions of these two plasmids. If the cloned insert is not in the proper translational reading frame to produce a hybrid protein, modifications to shift it inframe can be carried out at other sites in the polylinker. Thus, on plates containing XGal and IPTG, colonies of JM83 cells containing the vector alone are blue while recombinants are white. The malE region on pCG150 lacks a signal sequence, and is designed to produce the hybrid protein in the cyto-

activity without

requiring

a-complementation)

was

constructed as follows. Plasmid pCG150 was cleaved with BamHI + PvuII and a 1.2-kb DNA fragment was purified by gel electrophoresis. This fragment contains the lac promoter and the lacZ translation start fused to codons 28-392 of the malE gene (mature MBP is coded for by codons 27-396). This fragment was inserted into plasmid pMLB 1034 (Silhavy et al., 1984) that had been cleaved with EcoRI, tilled in with PolIk and dNTPs, then cleaved with BamHI. pMLB1034 contains the whole ZacZ gene except for the- promoter and the first eight codons. The resulting plasmid, pCG325, codes for an in-frame malE: : ZacZ-coded hybrid protein that produces P-galactosidase activity without a-complementation (Fig. 2). (c) Construction

of the maZE:: PstI endonuclease

gene fusion The maIE: : PstI endonuclease gene fusion was constructed as illustrated in Fig. 3. The PstI restriction system is comprised of two divergently transcribed genes, one coding for the endonuclease PstI and the other for the modification methylase (M *PstI). It has been cloned on a 4-kb DNA fragment inserted into the Hind111 site of pBR322 (pGW4400, provided by G. Wilson; also see Walder was excised with et al., 1984). This fragment HindIII, purified, circularized by ligation with T4 DNA ligase, and then cleaved with HincII to invert the two divergent genes and to separate the PstI endonuclease gene from its promoter. The fragment was then inserted into the HincII site of pUC18, creating plasmid pCG228. A BamHI-Hind111 fragment containing the endonuclease gene without the promoter and the first eight codons was cut from pCG228, purified, and then inserted into pCG150 and pCG806 in place of the small BamHI-Hind111

pCG150 y%oRI~

y&c11

[--KpIfiSmal~

~SUCI-J

rK@Il

. . . ATGACCATGATTACGAATTCGAGCTCGGTACCCGGGCGAGCTCGGTACCCA

TTCGAAGAA --PstI-,

rBamHIl

mdE sequence ------------

[- ,SphI 1

rHindIII

rXbuI-~

r.SalI

1

r

GCGCAGACTGGGGATCCTCTAGAGTCGACC

1

CacZ

TGC AGG CAT GCA AGC TTG -

. . .

pCG806 CAGACTAATTCGAGCTCGGTACCC

. malE sequence-

.

~BamHI1

r X&z17 r--- SalI -J r- PstI --q rSphI~

rHindII1

1

GGG GAT CCT CTA GAG TCG ACC TGC AGG CAT GCA AGC TTG w Fig. 1. Structure restriction

of MBP fusion vectors.

sites present

as described

by Maniatis

(Yanisch-Perron

EcoRI-J$i&I

is the 1.2-kb Hid portion

et al. (1982); restriction

it is the normal

(filled-in) fragment fragment

1977). Double-stranded pCGI50,

enzymes

All plasmids

translation

initiation

corresponds

the signal sequence

of the polylinker

regions.

DNA was used as a template.

37.5-383 (the region of malE just preceding to the 392nd codon

Ap-resistance

of malE. For pCG806,

lac promoter.

Both plasmids

To sequence

manuscript sequencing

including

are based

on pUC18 translation

The malE region of pCG150

out as described

(Sanger

the gene fusion joint region, a primer corresponding

and LeZcr) was used (provided MBP),

by New England

et al., to the

Biolabs).

For

and the ACT codon in bold print

the ACT codon in bold print once again refers to the 392nd codon that the gene is truncated

is the

the malE region ofpCG806

see Duplay et al., 1984). The lower

was carried

aa of mature

the

DNA techniques

this site is the lacZa

in preparation);

of malE (C. Lee, unpubl.;

Nucleotide

the polylinker

before or after the name of a gene indicates

gene; P,,,

Biolabs.

sites; on pCG150

. .

and pCG806,

using recombinant

start followed by the mdE signal sequence.

the GAA codon in bold print is the 28th codon of m&E (the second

A prime notation

of pCG150

were constructed

were from New England

from pPL-5A (P.L., H.I. and J. Beckwith,

from pmalE, and includes

plasmid

of the figure shows the structure

circles represent

m&E translation

of the figure shows the sequence

maZE codons

The upper portion

regions of these plasmids.

et al., 1985). The blackened

start and on pCG806 l.l-kb

in the polylinker

k&5?

at the N or C terminus,

of malE.

respectively.

Ap,

25

methylase,

codonof malE

t

strain

was cotransformed nuclease pCG1001, methylase

CG864

(RR1 AmaZE Zen: : TnlO)

with a plasmid bearing the endo-

gene (either pCG410

or pCG828)

and with

a plasmid bearing the modification gene. pCGlOO1 is a pACYC184-based

plasmid (Chang and Cohen, 1978), and is compatible with the pUC18-based

\ +o plylinker

2-3 “4

0

.P

plylinker

.

pPL-6A’

% 0 %

. -----

PstI endonuclease

(4 MA ss: : malE: :phoA gene

plasmids.

fusion

AphoA ss : : maZE: :phoA fusion was constructed to test whether MBP hybrid proteins could be directed to the periplasmic space. The structure of the plasmid bearing this fusion, pPL-6A’, is shown in Fig. 2. The details of this construction will be presented elsewhere (P.L., H.I. and J. Beckwith, manuscript in preparation). As described in section e, below, and in Table II, thephoA,, signal peptide could direct the hybrid protein to the periplasmic space. Although thephoA signal sequence was used in this example, the generalized vector pCG806 was constructed with the maZE signal sequence. It is anticipated that the MBP signal peptide would direct hybrid proteins to the periplasm in the same manner.

K”

Fig. 2. Structure

of pCG325

the figure shows constructed pCG150

by inserting between

pMLB1034

28-392.

This plasmid

the 1.2-kb PvuII-BamHI

the EcoRI

shows the structure

plasmid

The upper portion

of pCG325.

fragment

(filled in) and BamHI

(Silhavy et al., 1984). The lower portion

of this plasmid Beckwith,

and pPL-6A’.

the structure

of pPL-6A’.

is identical

elsewhere

in preparation). to that

of pCG325,

The region 5’ to ma/E contains

the first 21 codons

ofphoA (phoA,,),

phatase

signal

contains

most of the remainder

(corresponding This plasmid

peptide.

1985). Solid and dashed the indicated

Solid circles

represent

notations

are as in Fig. 1.

and

3’ to the malE sequence alkaline

from pHI28

genes; arrowheads

scription.

in this codons

the alkaline phos-

lines indicate

indicate

at codon 33 phosphatase).

(H.I., unpublished),

and pCH2 (Hoffman

concentric

and J.

the phoA promoter

of phoA, starting

to the 12th aa of mature was constructed

(P.L., HI. comprising

encoding

The region

pm&E (C. Lee., unpublished)

of the figure

The malE sequence

and Wright, the extent of

the direction

translation

start

(e) Purification

of the hybrid proteins by affinity

chromatography

from

sites of

The details of the construction

will be presented

manuscript

of

was

of tran-

sites. Other

fragment. The resulting plasmids, pCG410 and pCG828, respectively, contain in-frame m&E : : PstI endonuclease gene fusions. Since the PstI restriction gene is lethal to E. coli K-12 in the absence its cognate

The MBP-/3-galactosidase hybrid protein was purified from SF1362[pCG325] by cross-linked amylose affinity chromatography as described in MATERIALS AND METHODS, section d. Crude extract from 1 liter of cells contained approx. 350 mg total protein and 760 000 units of /?-galactosidase activity (Miller, 1972). Using a theoretical specific activity for the hybrid protein of 225 000 units/mg (the specific activity of native /?-galactosidase corrected for the M, of the hybrid protein), it is estimated that the hybrid protein represents about 1% of the total protein. More than 70% of the fi-galactosidase enzymatic activity present in the crude extracts bound to the cross-linked column and was eluted with maltose buffer. This fraction contained 540 000 units of /I-galactosidase, and SDS-polyacrylamide gel analysis (see below) revealed that 90% of the protein was the size predicted for the hybrid protein. The /I-galactosidase activity that did not bind to the column could not be accounted for by a limitation of the capacity of the column; when this fraction was

26

r( Hind111

HindIII

Hind111 purified fragment

1

t Him11 T4Ligase

I

HincII

HincII

Ap

HincII

.-

Fig. 3. Construction in RESULTS

of MBP::PstI

(MBP-endonuclease)

AND DISCUSSION,

The solid circles represent

translation

coding region. The Hind111 fragment

fusion plasmids.

Details of the construction

section c. M and R refer to the PstI modification start sites, with the dashed ofpGW4400

arrows

was purified, circularized

indicating

methylase the direction

of pCG410

and endonuclease of transcription

and pCG828

are given

genes, respectively. and the extent of the

by ligation with T4 DNA ligase, and then cleaved with HincII

21

TABLE II Localization of alkaline phosphatase activity produced by the phoA,:

:malE: :phoA fusion

Alkaline phosphatase activitya

Cold osmotic shockb Released from spheroplasts”

In periplasm

In cytoplasm + membranes

% periplasmic

0.22 0.13

0.028 0.021

89 86

a Alkaline phosphatase activity was assayed as described by Brickman and Beckwith (1975). 2.5-ml cultures of MZ20[pPL-6A’] were harvested and washed as described, and the alkaline phosphatase assays were performed on 10 pl of 200 ~1 periplasmic and cellular fractions. Activity is expressed as the change in A,,,/min. b Periplasmic and shocked-cell fractions were prepared as described by Neu and Heppel (1965). c Periplasmic and spheroplast fractions were prepared as described by Neu and Heppel(1964).

all of the b-galactosidase activity still passed through the column. The fraction of the fi-galactosidase activity that originally was eluted with maltose buffer was dialyzed against 10 mM Tris * HCl, pH 7.2, and applied to a fresh column; all of the /I-galactosidase activity once again bound to the column and could be eluted with maltose buffer. These results suggest that the /I-galactosidase activity present in the flow-through represents a fraction of the hybrid protein that is cleaved to give an active /I-galactosidase fragment lacking the maltose-binding activity. The MBP-PstI fusion protein was purified as follows. The crude extracts from strains containing pCG410 and pCG828 were applied to a cross-linked amylose column and chromatography was performed as described in MATERIALS AND METHODS,

ases (not shown). From 1 liter of culture, approx. 100000 units of PstI activity could reproducibly be recovered from the affinity purification. Since native PstI endonuclease exists as a dimer, and the MBPPstI hybrid protein appears to be cleaved at the joint

section d. In both cases, 90-95 y0 of the total restriction enzyme activity in the crude extracts passed through the column. The remaining 5-10% of the PstI activity could be eluted from the column by maltose buffer. As judged by restriction assays using 1 DNA, the protein that was purified from the amylose column had the same site specificity as native PstI, and was free from contaminating nucle-

d. As shown in Table II, SS-90% of the alkaline phosphatase activity produced was present in the periplasm; of this fraction, 90 y0 of the activity was retained on the amylose column and eluted with maltose buffer. This demonstrates that the presence of a signal peptide can direct this hybrid protein to the periplasmic space.

applied

to a fresh amylose column,

region

in

ViVO

(See

RESULTS

AND

DISCUSSION,

section f), it is not known whether the hybrid protein accounts for all of this endonuclease activity; for example, some of the activity could arise from mixed dimers of the hybrid protein and the PstI portion of the cleaved hybrid. The hybrid protein produced by the phoA,,: : maIE: :phoA fusion was purified as follows. The osmotic shock fluid from cells bearing pPL-6A’ was dialyzed against 10 mM Tris +HCl, pH 7.2, and applied to a cross-linked amylose column, and chromatography was performed as described in MATERIALS AND METHODS, section

to invert the two divergent genes and to separate the PstI endonuclease gene from its promoter. The fragment was then inserted into the HincII site ofpUC18, creating plasmid pCG228. A BarnHI-Hind111 fragment containing the endonuclease gene without the promoter and the first eight codons was cut from pCG228, purified, and then inserted into pCGl50 and pCG806 in place of the small BamHI-Hind111 fragment. The resulting plasmids, pCG410 and pCG828, respectively, contain in-frame malE_PstI endonuclease gene fusions. pCG410 lacks the malE signal sequence (‘malE’) while pCG828 includes the normal malE translation start and signal sequence (malE’).

28

gel anal-

ysis of the hybrid proteins

scribed in MATERIALS AND METHODS, section f, and in Fig. 4. For the MBP-P-galactosidase hybrid protein, the major band on the gel had an apparent M,

The fractions that were eluted with maltose buffer were pooled, concentrated and analyzed by SDS-polyacrylamide gel electrophoresis as de-

of 156 x 103, as predicted by the sequence of the gene fusion. Minor bands, which are presumed to be degradation products of the hybrid protein, were also

(f) Sodium dodecyl sulfate-polyacrylamide

205K 205K 116K 97.4K

116K 97.4K

45K

45K

29K 29K

B

A Fig. 4. SDS-polyacrylamide acrylamide Centricon

g&l electrophoresis microconcentrators

gel analysis of MBP$-galactosidase as described (Amicon)

(Laemmli,

according

to the gel. Gels were run at 30 mA (constant MW-SDS-200) B (97.4 kDa), phoresis

carried

were bovine carbonic b-galactosidase

(116 kDa),

out on protein fractions

eluted from the cross-linked 1, purified MBP.

protein

instructions.

and stained with Coomassie

and myosin

(205 kDa).

from cells bearing

polyacrylamide

eluted from the cross-linked

Proteins

were concentrated A 5-lo-pg

brilliant

were analysed lo-20

by SDS-poly-

x by ultrafiltration

sample of each protein

blue. Proteins

in

was applied

used as M, markers

(Sigma

(29 kDa), egg albumin (45 kDa), bovine serum albumin (66 kDa), rabbit phosphorylase pCG325.

(K = kDa). Lanes:

gel electrophoresis amylose

(Panel A) 0.1 y0 SDS-7%

1, M, markers;

amylose column with maltose buffer; 4, flow-through

extract. (Panel B)0.1 y0 SDS-lo%

hybrid proteins.

to be analyzed

to the manufacturer’s

current)

anhydrase

and MBP-PstI

1970). Samples

from the cross-linked

carried out on protein fractions

column with maltose

polyacrylamide

gel electro-

2, purified MBP; 3, purified hybrid protein

buffer; 2, purified

amylose column;

5, crude cellular

Tom cells bearing pCG410.

native PstI endonuclease;

Lanes:

3, purified

29

present.

HPLC

fraction

indicated

retained

fl-galactosidase

SDS-gel hybrid

separation that

analysis

protein

signal sequence) to that predicted

156-kDa

species

of maltose-eluted by PCG410

showed three bands.

a

The top-most

A4, of 78 x 103, corresponding The

two lower bands corresponded to 40 kDa and 45 kDa (mature MBP runs at about 40 kDa), and comprised 90% of the total protein in the maltoseeluted fraction. This indicates

that the hybrid protein

is sensitive to one or more host proteases, which cleave with some specificity at the joint region of the fusion. The identical analysis with the maltose-eluted fraction prepared from cells bearing pCG828 (with a malE signal sequence) showed three bands in the same relative positions, with the position of the two lower bands indicating a slightly higher M, (not shown). Only 20 y0 of the PstI enzymatic activity was released in the shock fluid of cells bearing pCG828, indicating that the hybrid protein was not efficiently secreted into the periplasmic space. (g) Immunological

in turn facilitate

This purification

MBP-PstI (lacking

from the gene fusion sequence.

using the vectors pCG1.50 and pCG806;

these fusions hybrid protein

activity (not shown).

produced

band had a apparent

to construct

of the maltose-eluted

only the

analysis of the hybrid proteins

Two-dimensional immunodiffusion analysis was performed on the maltose-eluted fraction from cells bearing pCG325. This fraction formed a single precipitin line both with antiserum directed against MBP and with antiserum directed against /?-galactosidase (not shown). Native P-galactosidase formed a precipitin line only with the anti+galactosidase antiserum and native MBP formed a precipitin line only with the anti-MBP antiserum. Monoclonal antibody directed against PstI strongly inhibited the activity of both the native PstI enzyme and the MBP-PstI hybrid protein (not shown). Remarkably, the antiMBP antiserum also inhibited the restriction activity of the MBP-PstI hybrid protein present in the maltose-eluted fraction. This inhibition could be released by adding excess MBP, whereas the inhibition by anti&t1 monoclonal antibody could not. (h) Conclusions The technique outlined here is a simple and powerful tool for expressing and purifying foreign peptides in a hybrid protein form. Fusions to MBP are easy

by binding

the purification

to cross-linked

of a

amylose.

gives a high yield and degree of

purity in a single step, carried out under nondenaturing conditions at neutral pH. The procedure can be used in a small-scale

purification,

for example

the expression and purification of a target protein suitable for preparing antisera. It can also be easily adapted to a large-scale purification of a commercially valuable

protein.

In addition,

fusions

con-

structed with pCG806 produce a hybrid protein containing the MBP signal sequence; the results with the PhoA,,-MBP-PhoA fusion indicate that in at least some cases, a signal peptide can direct an MBP fusion protein to the periplasm (see RESULTS AND DISCUSSION, section

d and e, and Table II).

Two drawbacks of the approach as described here are: (i) the stability of the hybrid protein in vivo is variable and hard to predict, as illustrated by the examples presented in this study, and (ii) the hybrid protein includes a carrier peptide (MBP) which may not be acceptable in some applications. ,Possible solutions to these problems are now being investigated.

ACKNOWLEDGEMENTS

This work was supported by a grant from New England Biolabs. We would like to thank L. Moran and B. Slatko for performing the nucleotide sequencing, and Ira Schildkraut, Elisabeth Raleigh and Jon Beckwith for helpful discussion. This paper is dedicated by C.G., P.L. and P.D.R. to the memory of Hiroshi Inouye, who died on July 24, 1986.

REFERENCES Brickman, E. and Beckwith, J.: Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and $80 transducing phages. J. Mol. Biol. 96 (1975) 307-3 16. Chang, A.C.Y. and Cohen, S.N.: Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacterial. 134 (1978) 1141-l 156.

30

Duplay,

P., Bedouelle,

Hofnung,

H., Fowler, A., Zabin,

the maltose-binding

I., Saurin, W. and

protein

of Escherichia coli K-12. J. Biol.

Chem. 259 (1984) 10606-10613. Ferenci, T. and Klotz, U.: Afftnity chromatographic the periplasmic

maltose-binding

of

FEBS Lett. 94 (1978) 213-217. Hoffman,

C.S. and Wright,

alkaline phosphatase:

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