Gene fusion vectors based on the gene for staphylococcal protein A

369

Gene, 23 (1983) 369-378 Elsevier GEN 00810

Gene fusion vectors based on the gene for staphylococcal protein A (Recombinant

Mathias

DNA;

hybrid

protein;

IgG-binding;

/3-galactosidase;

Uhl&n a,b, Bjiirn Nilsson a, Bengt Guss b, Martin

Escherichia

coli; plasmids)

Lindberg b, Sten Gatenbeck a and Lennart

Philipson b,* a Department of Biochemistry, Royal Institute of Technology, S- 100 44 Stockholm, Tel. 8- 7877513, and b Department of Microbiology, University of Uppsala, The Biomedical Center, Box 581, S-751 23 Uppsala (Sweden) Tel. IS-174584 (Received March 3rd. 1983) (Revision received April 13th, 1983) (Accepted April 14th, 1983

SUMMARY

Two plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusion, have been constructed. These vectors will allow fusion of any gene to the protein A gene: thus giving hybrid proteins which can be purified, in a one-step procedure, by IgG affinity chromatography. As an example of the practical use of such vectors, the protein A gene has been fused to the 1ac.Z gene of Escherichia coli. E. coli strains containing such plasmids produce hybrid proteins with both IgG binding and P-galactosidase activities. The hybrid moiety with high efficiency without losing competitive elution with pure protein A. which means that the in vivo product can

protein(s) can be immobilized on IgG-Sepharose by its protein A its enzymatic activity and they can be eluted from the column by The fused protein(s) also binds to IgG-coated microtiter wells be used as an enzyme conjugate in ELISA tests.

INTRODUCTION

Gene fusion in which the coding sequence of two or more genes are spliced together by genetic

* Present address: European Molecular Biology Laboratory, Postfach

1022.09, D-6900

Heidelberg

1 (F.R.G.)

Tel. 6221-

387200. Abbreviations: METHODS,

bp, base pairs; ELISA, see MATERIALS

AND

section c; Fc, constant part of immunoglobulins;

IgG, immunoglobulin

type G; kb, kilobase pairs; lacZ’, trun-

cated 1acZ gene; ONPG, o-nitrophenyl P-D-galactoside; phosphate-buffered

PBST,

saline with 0.05% Tween 20; SDS, sodium

dodecyl sulfate; Xgal, 5-bromo-4-chloro-3-indolyl-B-D-galactoside. 0378-I 119/83/$03.00

0 1983 Elsevier Science Publishers B.V.

approaches is a technique of growing importance. It has been used to study protein transport mechanisms (Silhavy et al., 1977), plasmid replication (Light and Molin, 1982), and gene expression (Zabeau and Stanley, 1982). In particular, the lucZ gene of E. coli coding for the enzyme P-galactosidase (P-D-galactosidase galactohydrolase, EC 3.2.1.23) has been preferred as the paternal gene for constructing fused products (Casadaban et al., 1980). We have earlier reported on the cloning of the gene coding for staphylococcal protein A in E. coli (Liifdahl et al., 1983). This protein is used as an immunological tool due to its specific binding to

the Fc part of immunoglobulins

of many

species

by sonication

including man (MacSween et al., 1981). This property is used for isolation of IgG by selective affin-

buffer

ity chromatography involving covalently coupling of protein A to Sepharose 4B (Pharmacia Fine

supernatants

Chemicals). Protein using IgG-Sepharose two plasmid have

been

genes

expressed affinity

vectors,

@PA11

constructed

to the region

encodes

A can likewise be isolated by columns. We describe here, and pSPA12,

to allow

of the protein

its IgG binding

capacity.

after the fusion chromatography.

fusion

which

of other

A gene which Hybrid

can be purified The usefulness

proteins by IgG of these

vectors is exemplified in this paper by manufacturing protein A-~“galactosidase hybrid proteins.

MATERIALS AND METHODS

(a) Bacterial strains and plasmids E. cob strains HBlOl (Boyer and RoullandDussoix, 1969), XAC (Miller et al., 1977), and RR1 delM15 (Langley et al,, 1975) were used as bacterial hosts. The plasmid vectors were pBR322 (Bolivar et al., 1977), pTR262 (Roberts et al., 1981), and pSKS104 and pSKS106 (Casadaban, M.J., Martinez-Alas, A., Shapira, SK., and Chou, J., personal communication). Phage vector Ml3mp8 was obtained from New England Biolabs, Beverly, MA, USA. (b) DNA constructions Restriction endonucleases, T4 DNA ligase (New England Biolabs) and alkaline phosphatase (Sigma, St. Louis) were used according to the suppliers’ recommendations. Transformation of E. coli was made according to Morrison (1979). Plasmid DNA was prepared according to Tanaka and Weisblum (1975). For scoring large numbers of clones for plasmid DNA an alkaline extraction method was used (Bimboim and Doly, 1979). (c) Enzymatic assays R~ombinants cont~n~g a functio~ai IucZ gene were scored by plating on X-gal plates as’ described by Miller (1972). Cell extracts were made

(3 X 30 s in an MSE-sonicator)

contai~ng

protease

in~bitors

in a

(Persson

et

al., 1979). The cell debris were spun down and the saved

quantitation

of protein

enzyme-linked described was

for

Detection

A was performed

immunosorbent

by Lofdahl

assayed

analysis.

and by an

assay (ELISA)

as

et al. (1983). /3-galactosidase

by a calorimetric

ONPG (Sigma product as described by Miller

procedure

using

No. N-1127) as substrate (1972) with the following

modifications. The assay was performed at + 8°C and the activity was measured at 405 nm. One unit represents

a change of one A unit at 405 nm/min.

P-Galactosidase activities of the fused proteins coupled to IgG~Sepharose were determined at i- 8°C by rocking the tubes to prevent sedimentation. (d) Binding and elution of protein A Binding of protein A to IgG-Sepharose (Pharmacia, Sweden) was performed by rocking crude lysates with 0.1 vol of sedimented gel for 1 h at f 8°C. Elution of protein A from IgG-Sepharose columns was performed as described by Lofdahl et al. (1983) using a glycine buffer, pH 3.0. Since /3galactosidase is inactivated at this low pH, pure protein A was used to competetively release the in vivo fused protein A+gaIactosidase. 50 ~1 sedimented gel was eluted with 2 X 200 ~1 of PBST containing different amounts of pure protein A. Retained materials on the IgG columns were analysed by SDS-polyac~la~de gels as described previously (Lofdahl et al., 1983).

RESULTS

(a) DNA sequence of the protein A gene To make a fusion between two genes, it is desirable to know the DNA sequence and its deduced amino acid sequence around the fusion point, to align the reading frames in both genes and thus obtain a functions hybrid protein. The sequence of the whole gene coding for staphylococcal protein A, has been determined (Uhlen, M.

371

A.

iSI 181

I

E 289

I

D

457

A

I\-------

640

814

988

1162

1630

Sau3A

TaqI

6ClI

B. 1

Sau3A

I

355

I

487

PstI

Sau3A

Hindm

I

BclI

EcoRY

1572

1770

I

735

1096

1541

391

C.

protein

D.

P-galactosidase

A

protein

Fig. 1. Map of protein is a signal sequence, lacks IgG-binding corresponding pSPA13;

A

A and its fusion products.

A-D activity.

are IgG-binding

I @-galactosidase

(A) Schematic

regions,

Note that &/I

drawing

of the gene coding for protein

E is a region homologous

The size is given in bp starting

DNA sequence.

pSPA13

to A-D,

A with its different

and X is the C-terminal

at the TuqI site ( = 1) (see B). (B) Detailed

sites also are Sau3A

sites. (C and D) Schematic

PSPA

drawings

regions.

part of protein restriction

14

map

S

A which of the

of gene fusion plasmids.

(C)

(D) pSPAl4.

and GUS, B., manuscript in preparation) schematically shown in Fig. 1A. Based

and is on this

sequence we constructed two gene fusion vectors by inserting multilinkers from M13mpS into the Sau3A and the PstI sites at nucleotides 1096 and 154 1, respectively (Fig. 1B). These sites are situated before and after the repetitive part of region X which is thought to mediate binding of protein A to the peptidoglycan of the cell wall in Staphylococcus aweus (Sjiidahl, 1977a). The DNA sequences and the deduced amino acid sequences around these restriction sites are shown in Fig. 2.

1069

1081

1513

1525

(b) Construction of fusion vectors To construct the fusion vectors, various restriction fragments of the protein A gene were subcloned into E. coli. These steps are schematically shown in Fig. 3. A 2.1-kb EcoRV fragment, containing the whole protein A gene, was purified by sucrose-gradient centrifugation. The purified fragment was cut with 7’aqI and the fragment spanning from nucleotides 1 to 1770 in Fig. 1B was inserted between the ClaI and EcoRV sites in pBR322. The resulting plasmid, pSPA8, contains

1093 1105 1117 I I 1 Sau3A 1 I AACGGCTTCATC CAAAGCCTTAAA GACECTTCG GTGAGCAAAGAA ATTTTAGCAGAA AsnGlyPhe Ile GlnSerLeuLys AspAspProSer ValSerLysGlu 11eLeuAlaGlu

A.

B.

I

PstI '547

1561

I

GCAAACGGCACT ACTGCTGACAAA ATTGGAT AACAAATTGGCT GATAAAAACATG AlaAsnGlyThr ThrAlaAspLys IleAlaAlaAsp AsnLysLeuAla AspLysAsnMet

Fig. 2. The nucleotide deduced

1537

I

I

sequence

amino acid sequences

around

the Sau3A

are also shown.

site at position

1096 (A) and the PstI site at position

1541 (B) (see Fig. 1B). The

372

“indm

andP,tt

Fig, 3. Schematic presentation of constructions of plasmids containing the whole or parts of the protein A gene. A few restriction sites are indicated. Boxes show the relative positions of genes coding for protein A (PROT A), &lactamase (TET) and the N-terminal part of &galactosidase

(AMP). tetracycline resistance

(LAC Z’). The arrows indicate the orientation of the genes: the replication origin is

marked Ori.

the whole structural gene of protein A preceded by the protein A promoter, and lacks any E. coli promoter before the inserted gene. The gene is still expressed in E. cc& (Table I) confirming the data from Lbfdahl et al. (1983) that the staphyloco~al promoter is recognized by E. coli RNA polymerase. Another plasmid was constructed in which a fragment of the protein A gene was inserted into the plasmid pUR222 (Rtither et al., 1981) at its BamHI site which is located in direct proximity to

a single EcoRI site. The latter site now becomes suitable for gene fusions. Since the plasmid pUR222 contains a fragment of the Iad gene, a biological assay could be used to detect successful recombinants. A schematic presentation of the steps involved in the construction of this plasmid, pSPA10, is shown in Fig. 3. The 2.1-kb fragment purified by sucrose-gradient centrifugation was cut with Sau3A. The digest was run on a preparative 8% polyacrylamide gel and the DNA fragment of approx. 600 bp was cut out, eluted and purified.

373

striction

TABLE

I

Protein

A content

of E. coli hc-

cells containing

different

enzymes

previously

Hind111 and &I.

described

by

Lofdahl

The plasmid et al. (1983)

plasmids

pSPA2,

E. coli XAC cells were grown

the two digests were mixed (see Fig. 3). After ligation the DNA was transformed into E. coli

the protein

A determined

METHODS, lysates;

section

E. Total column

in MATERIALS

is the concentration

eluate is the measured

to an IgG Sepharose buffer

to A,,,, = 1.0, disintegrated,

as described

concentration and elution

and AND

in crude

after adsorption with 0.1 M glycine

pH 3.0. Protein A content

Plasmid

0

0

16

16

pSKS106 pSPA8 pSPA13

4

4

pSPA14

1

1

Blue colonies were isolated one of these was investigated plasmid,

fused to the Sau3A Since

contained

site at position

the P-galactosidase

the 5’ gene

1096 (Fig. 1B).

in the hybrid

gene

of

Ala Ala Gly Arg Arg Ile Pro Gly Asn GCT GCA GGT CGA CC& ATC CCC GGG AAT TC -PSt.1

Sal XmaI

pSPAl0

pSPA10, only codes for the a-fragment, detectable enzymatic activity is dependent on complementation from a chromosomal gene product (Miller, 1972). In the strain RR1 this complementation functions in vivo but we have not been able to detect complementing activity in cell-free lysates. To construct a fusion vector, a DNA linker containing multiple restriction sites was inserted at the EcoRI site of pSPAl0. Phage vector M13mp8 and

ASP Ser Arg Gly SW Val Asp tell Gin AAT TCC CGG GGA TCC GTC GAC CTG CAG -PstI BamHI ECORI xr

and the DNA from by restriction analy-

gene and had the lad’

end of the protein-A

This fragment corresponds to the part of the protein A gene between nucleotides 487 and 1096. The DNA was mixed with BamHI digested DNA from pUR222. After ligation and phenol extractions, the whole DNA mixture was cut with re-

and

RR1 cells. Transformants were selected on Xgal plates containing both tetracycline and ampicillin.

sis. This hybrid

(pg/ml) Eluate

Total

was also cut with the same enzymes

BamHI Sal

AccI

ACCI

EcoRI SmaI XmaI

Pst

Hind

III “h

A. Fig. 4. Schematic the deduced (B) pSPA12.

drawing

of two vector plasmids

amino acid sequence

adapted

at the Ml3 multi-linker

for fusions. The abbreviations junction

are as in Fig. 3. The nucleotide

point are shown. The plasmids

are not drawn

sequence

and

to scale. (A) pSPAl1;

374

plasmid pSPAl0 were cut separately with EcoRI and PstI, mixed, ligated and transformed into E. coli HB 10 1. An ampicillin-resistant, tetracyclinesensitive

clone

was

analysed

and

PstI 1

a schematic

drawing of its plasmid called pSPAl1, is shown in Fig. 4A. The deduced amino acid sequence across the

fusion

reading

point

provides

a guide

for

aligning

frames after gene fusion. EcoRI

(c) Fusions to the 1ac.Z gene

t

B.

Fig. 5. Schematic

A number of gene fusion vectors containing the 1acZ gene have been constructed by Casadaban et

between

drawing

genes coding

abbreviations not drawn

of two plasmids

for protein

containing

part of /3-galactosidase.

to scale. (A) pSPA13:

fusions

A and P-galactosidase.

are as in Fig. 3. LacZ specifies

the carboxy-terminal

al. (see MATERIALS AND METHODS, section a). These vectors encode an enzymatically active P-galactosidase fragment lacking a few amino acids at the N-terminus. One of these plasmids, pSKS104, was digested with EcoRI and mixed with EcoRI-digested pSPAl1. After ligation, the DNA was used to transform strain E. coli XAC lac-. Approximately half of the tetracycline and ampicillin-re-

-Pstl

Pstl

A.

The

DNA coding

for

The plasmids

are

(B) pSPA14.

sistant clones were light blue on Xgal plates. A restriction map of the plasmid from one of these clones is shown in Fig. 5A. The gene fusion is shown in Fig. 1C and the deduced amino acid sequence over the fusion point is presented in Fig.

SmaI A.

Sau3A EcoRI BamHI Sal1 PstI Hind111 ---...AAAGACGATCCGGGGAATTCCCGGGGATCCGTCGACCTGCAGCCAAGCTTGGCACTGGCC... LysAspAspProGlyAsnSerArgGlySerValAspLeuGlnProSerLeuAlaLeuAla <

I

protein A

pUR222 linker

,6'7 I

I

I

I

8

1aZ' pSKS104

Ml3 mp8 multi-linker

SmaI PstI --I

B.

Sal1 BamHI

EcoRI

...4AAATTGCTGCAGGTCGACGGATCCCCGGGAATTCACTGGCC... LysIl eAlaAlaGlyArgArgIleProGlyAsnSerLeuAla < protein A Fig. 6. The nucleotide pSPA14

(see Fig. 5).

,6

I

7

8

I

I

Ill13mp8 multi-linker sequence,

the restriction

sites and the deduced

1acZ' pSKS106 amino acid sequences

around

the fusion sites. (A) pSPA13;

(B)

315

6A. The gene consists of the protein A gene down to the Sau3A site at position 1096, a multilinker

TABLE

consisting of 44 nucleotides and the IacZ gene coding for P-galactosidase from amino acid 7.

microtiter

Plasmid pSPA2 (Fig. 3) contains a unique PstI site at position 1541 of the protein A gene (Fig.

mids were disintegrated

1B). This plasrnid

coated

microtiter

lysate

and

was therefore

the IucZ gene of pSKS106. digested with PstI, mixed, transform

E. coli XAC

50% of the

ampicillin

used for fusion

to

Both plasmids were ligated and used to

lac-

cells. Again

and

tetracycline-resistant

approx.

clones were found to be Lac+ on Xgal plates. The resulting plasmid pSPA 14, is shown schematically in Figure 5B and the gene fusion is also outlined in Figures 1D and 6B. By cutting these plasmids with EcoRI and religating, plasmid pSPA12 was obtained. This plasmid is shown in Figure 4B. The gene consists of the protein A gene down to the PstI site at position 1541, a multilinker consisting of 21 bp and the IacZ gene coding tosidase from amino acid 7.

for /3-galac-

(d) Detection and quantitation of protein A from E. coli clones Cell extracts of E. coli XAC lac- cells containing four different plasmids were prepared as discussed in MATERIALS AND METHODS, section c. The plasmids were pSPA8, containing the whole protein A gene, pSKS106 with the P-galactosidase gene, and pSPA13 and pSPA14 containing different protein-A-P-galactosidase gene fusions. IgGSepharose affinity chromatography was used to purify the protein A moiety from the cell extracts. The proteins which bound to the column were eluted with 0.1 M glycine pH 3.0. The protein A content before and after the affinity step was tested with an ELISA technique and the result is shown in Table I. All protein A was retained by the column and efficiently eluted. The total content of protein A is 1 pg/ml for pSPA14,4 pg/ml for pSPA13 and 16 pg/ml for pSPA8. Since P-galactosidase is irreversibly inactivated by the glycine buffer at pH 3.0 (unpublished results), the enzyme activity of the eluted proteins from the IgG-Sepharose could not be detected. Table II shows that the hybrid proteins from pSPA13 and pSPA14 also bind to IgG coated

II

P-Galactosidase

activity

after

immobilisation

E. coli XAC cells at A 55,, = 1.0 containing determined

MATERIALS Plasmid

to IgG-coated

wells.

in crude

and the activity lysates

and after immobilization

wells. Values

1 unit

of

AND

activity

METHODS,

Activity

the indicated

of /3-galactosidase

are calculated is defined

plaswas

to IgG-

per ml of cell as described

in

section c.

in

crude lysates

% activity immobilised

(units/ml) pSKS106

14.0

0

pSPA8

0.0

0

pSPA13

0.4

41

pSPA14

0.1

63

microtiter wells. Cell lysates were added to coated microtiter wells, allowing time for binding, and unbound proteins were washed off. Bound P-galactosidase was assayed by adding ONPG as substrate to the wells and between 40-80% of the activity was retained in the wells. (e) Biuding to IgG-Sepharose Lysates from 10 ml cell cpltures (A,,, = 1.0) containing plasmids, pSKS106, pSPA13 and pSPA14, were mixed with 1 ml of IgG-Sepharose. The mixtures were rocked at + 8°C for 1 h and the supernatants were collected. The sediments were washed four times with 10 ml PBST (phosphate buffered saline with 0.05% TWEEN 20) and the supernatants of the last washes were collected. The IgG-Sepharose was resuspended in PBST and aliquots were transferred to smaller tubes. The /3-galactosidase activities bound to the IgG-Sepharose and in the supernatants were measured. As shown in Table III, the P-galactosidase from cells containing pSKS106 (control) did not bind to IgG-Sepharose, as shown above for the IgG coated microtiter wells (Table II). In contrast, /3-galactosidase from cells expressing the hybrid proteins (pSPA13 and pSPA14) binds to the gel. Although only 2% of galactosidase is expressed compared to pSKS106, more than 70% of the P-galactosidase activity derived from the fusion plasmids is immobilized. Analysis of the fusion proteins re-

376

TABLE

umns

III

@-Galactosidase Crude

activity

lysates

after immobilization

of E. cali XAC

IgG-Sepharose

as described

ODS,

section

wash

and

immobilized AND

given as percent Plasmid

in MATERIALS

d. The activity

MATERIALS

cells were allowed

were

determined

METHODS,

section

to bind

AND

in the supernatant,

and PBST-buffer

cont~ning

pure protein

A

was added. Table IV shows that at least half of the

to lgG-Sepharose

activity

to

can be eluted.

METH-

in the third described

in

c; the values

as

are

DISCUSSION

of crude lysates.

% activity in

% activity

% activity

supernatant

in wash

immobilized

We have earlier described the isolation and characterization of the gene coding for staphylo-

84.9

0.1

0.2

pSPA13

3.0

0.3

71.6

coccal protein A (Liifdahl et al., 1983). The structural gene consists of three function~ly distinct

pSPA14

6.0

0.0

78.4

parts. The S-end

pSKS106

codes for a signal peptide

which

aids the transport through the cytoplasmic membsane both in S. aureus and E. coli. The second part is a stretch coding for five nearly homologous regions, named E, D, A, B and C, of which at least D, A, B and C are responsible for the binding to the Fc portion of many immunoglobulins (Sjiidahl, 1977b). At present we do not know whether region E binds to IgG (Ldfdahl et al., 1983). Finally, the 3’-end of the gene codes for a region X, which is attached to the peptidoglycan in S. uu~eus. The whole structural gene which codes for 516 amino acids (Uhlen, M. and Guss, B., manuscript in preparation) is shown schematically in Fig. 1A together with a physical map (Fig. 1B).

tained on the IgG columns by SDS-polyacrylamide gels (not shown) revealed that pSPA13 accumulated products of M, around 42000, and pSPA14 of around 60 000. Since the M,s of protein A moiety in these two plasmids are 30000 and 40~0, respectively, the majority of the fusion proteins are not full size but larger than the protein A moiety. The intact fusion protein is only present in minute amounts. The low yield of functional galactosidase may therefore depend on proteolysis or premature termination of translation.

This report describes the construction of two plasmid vectors, pSPAl1 and pSPA12 based on the protein A gene. A multilinker (M13mpX), containing multiple restriction sites, has been inserted at the Suu3A site at position 1096 and the PstI site at position 1541 (Fig. IB). These vectors (Fig. 4, A

(f) Elution of hybrid proteins from IgG-Sepharose As the P-galactosidase is inactivated by glycine buffer pH 3.0 we tried to elute the fusion protein by competing with pure protein A (Pharmacia Fine Chemicals). Aliquots of IgG-Sepharose with bound fusion proteins, were transferred to col-

TABLE

IV

Elution

of bound

P-galactosidase-protein

The values are given as percent protein

A. Immobilized

ALS AND METHODS, Elution

medium

No protein

10 mg protein

of immobilized

refers to the bound

activity

j&galactosidase

from IgG-Sepharose before

elution.

activity

A/ml A/ml

Elution

after elution.

medium

was PBST and different

Elution

was performed

amounts

as described

section d. pSPAl3

A

2.5 mg protein

A fusion protein

and B) are designed to aid fusion between the protein A gene and other genes by in vitro recom-

pSPA14

% activity

% activity

% activity

% activity

eluted

not eluted

eluted

not eluted

0

91

0

35

63

30

62

64

31

51

45

89

of pure

in MATERI-

bination duced terminal

techniques. with

IgG

protein

Hybrid

binding

proteins

capacity

A moiety.

Gene

will be produe to its Nfusion,

using

protein made

A-enzyme chemically

conjugates in vitro.

which

Table

more than 70% of both hybrid

III

proteins

have

been

shows

that

bind to the

vector pSPAl1, leads to hybrid proteins lacking region X but in pSPA12 most of region X is intact.

IgG-Sepharose while the /3-galactosidase (pSKSl06) fails to bind.

The plasmid pSPA12 will therefore yield a hybrid protein with region X as a “spacer” between the

The hybrid protein could be eluted from the affinity column by competing with pure protein A (Table IV). Competitive elution was used, because

IgG binding moiety and the second protein. The two vectors were used to fuse the protein gene to the truncated 1acZ genes zymatically active carboxy-terminal /3-galactosidase

(Casadaban

A

encoding enfragments of

et al., 1980). When the

two resulting plasmids, pSPA 13 and pSPA 14, were introduced into strain E. coli XAC lac-, the figalactosidase activity in cell free lysates was only 2% of the activity observed in cells containing the complete lac operon (pSKS106). It is at present unknown if the expression is restricted at the level of transcription, translation or post-translation. As the protein A gene is inefficiently expressed when inserted after the tet promoter of pBR322 (not shown) it is unlikely that the transcription step is limiting. However, the start codon TTG, which is present in the protein A gene (Lofdahl et al., 1983) has so far not been identified in Gram-negative bacteria. This codon may be poorly recognized by E. coli ribosomes leading to reduced translation. In addition, the N-terminal single peptide of hybrid proteins converts the P-galactosidase moiety to a membrane bound protein (data not shown and Silhavy et al., 1976), which could influence the overall rate of expression. This could also explain why cells containing gene fusions produce protein A in lower amounts than cells containing the intact gene pSPA8 (Table I). However, it is hard to explain why the fusion vector pSPA13 lacking region X gives 3-4 times more hybrid protein than the fusion vector pSPA14 containing this region (Tables I and II). Proteolysis of the hybrid protein or pretermination of translation is probably also involved as demonstrated by SDS-polyacrylamide gel analysis. The affinity of the gene fusion products to IgG, was tested by measuring the binding to IgG coated microtiter wells and to IgG-Sepharose. The hybrid proteins bind well to the microtiter wells and they can therefore be used in ELISA-tests instead of

buffers ordinarily used for releasing protein glycine pH 3.0, 3 M potassium thiocyanate M lithium versibly shown).

diiodosalicylate

inactivate However,

the inserted

(Langone,

control

A, i.e., or 0.1

1982)

irre-

the P-galactosidase (data not for other gene fusions, in which

protein

is not sensitive

to these buffers

any of these elution methods might be used. The techniques for immobilizing enzymes have been refined during the last decade (Bucke and Wiseman, 1981). For further development of these techniques, hybrid proteins containing the protein A moiety will be of great value. Enzymes fused to protein A in this way can be directly immobilized to an IgG affinity column. The specific affinity between IgG and protein A assures a one-step procedure giving a pure and immobilized enzyme. Another advantage compared to conventional immobilization techniques is that the affinity column can easily be regenerated by lowering the pH. The fused proteins are produced in relatively low quantities in E. coli. In order to improve the yield, we will try to insert the fused genes into a gram positive host vector system or into vectors containing promoters and other control elements necessary for high expression in E. coli.

ACKNOWLEDGEMENTS

This investigation was supported by grants from the Swedish National Board for Technical Development (M.U. and B.N.), the Swedish Medical Research Council (M.L.) and Pharmacia Fine Chemicals, Uppsala (M.L.). We are grateful to Drs. Kurt Nordstrom, John Sjiiquist and Marc Zabeau for critical comments and advice. We thank Hans-Olof Pettersson for skilful technical assistance and Christina Pellettieri for patient secretarial help.

378

REFERENCES

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H.C.

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J.: A rapid

alkaline

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Miller, J.H.:

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