Immunology Today, vol. 5, No. 8, 1984
244 18 Olsen, R. G., Hoover, E. A. and Schaller, j . P. et al. (1977) Cancer Res. 37, 2082-2085 19 Schaner, J. P., Hoover, E. A. and Olsen, R. G. (1977),]. Natl Cancerlnst. 59, 1441-1450 20 Hebebrand, L. C., Mathes, L. E. and Olsen, R. G. (1977) Cancer Res. 39, 443-447 21 Fowler, A. K., Twardzik, D. R. and Reed, C. D. etal. (1977) CancerRes. 37, 4532-4533 " 22 Mathes, L. E., Olsen, R. G. and Hebebrand, L. C. et aL (1978) Nature (London) 274, 687-689 23 Mathes, L. E., Olsen, R. G. and Hebebrand, L. C. et al. (1979) Cancer Res. 39, 950-955 24 Bolognesi, D. P., Montelaro, R. C. and Frank, H. et aL (1978) Science 199, 183-186 25 Denner, J., Wunderlich, V. and Bierwolf, D. (1980)Acta. BioL Med. Ger. 39, 19-26 26 Cianciolo, G.J., Matthews, T.J., Bolognesi, D, P. and Snyderman, R. (1980)J. Immunol. 124, 2900-2905 27 Cianciolo, G.J., Hunter, J , Silva, J. Haskill,J, S. and Snyderman, R. (1981),]. Clin. Invest. 68, 831-844 28 Stiff, M. I. and Olsen, R. G. (1983)J. Gen. ViroL 64, 957-959 29 Copelan, E. A., Rinehart, J . J . and Lewis, M. et al. (1983)J. Immunol. 131, 2017-2020 30 Wellman, M. L., Kociva, G.J. and Lewis, M. G. et aL (1984) Cancer Res. 44, 1527-1529 31 Zbar, B., Wepsic, H. T. and Rapp, H.J. etaL (1970)J. Natl. Cancerlnct. 44, 701-717 32 Snyderman, R., Altman, L. C., Hausman, M. S. and Mergenhagen, S. E. (1972)J. ImmunoL 108, 857-860 33 Boetcher, D. A. and Leonard, E..]. (1974)J. Natl GancerrL Inst. 52, 1091-1099 34 Hausman, M. S., Brosman, S. and Snyderman, R. et aL (1975).]. Natl Cancer Inst. 55, 1047-1054 35 Snyderman, R. and Stahl, C, (1975) in The Phagocytic Cell in Host Resistance (Bellanti, J. A. and Dayton, D. H., eds),'Raven Press, New York
36 Rubin, R. H., Cosimi, A. B. and Goetzl, E. J. (1976) Clin. Immunol. ImmunopathoL 6, 376-388 37 Snyderman, R., Seigler, H. and Meadows, L. (1977)J. Natl GancerInst. 58, 37-41 38 Snyderman, R., Meadows, L., Holder, W. and Wells, S. (1978)J. Natl Cancer Inst 60, 737-740 39 Snyderman, R. and Pike, M. C, (1976) Science 192, 370-372 40 Norman, S.J. and Sorkin, E. (1976),]. Nail Cancer Inst. 57, 135-140 41 Stevenson, M. M. and Mdtzer, M. S. (1976)J. Natl Cancer Inst. 57, 847-852 42 Nelson, M. and Nelson, D. S. (1978) Immunolog~ 34, 277-290 43 Spitalny, G. and North, R.J. (1977)J. Exp. Med. 145, 1264-1277 44 Pike, M. C. and Snyderman, R. (1976),]. Immunol. 117, 1243-1249 45 Cianeiolo, G. J., Herberman, R. B. and Snyderman, R. (1980),]. Natl Cancer Inst. 65, 829-834 46 Levy, M. H. and Wheelock, E. F. (1975),]. ImrnunoL 114, 962-965 47 Bendinelli, M., Toniolo, A. and Friedman, H. (1976) Ann. NYAcad. Sci. 276,431-441 48 Cianciolo, G.J., Lostrom, M. E., Tam, M. and Snyderman, R. (1983) ,jr. Exp. Meal 158, 885-900 49 Cianeiolo, G. J. and Snyderman, R. (1981)J. Clin. Invest. 67, 60-68 50 Cianeiolo, G.J., Phipps, D. and Snyderman, R. (1984)J. Exp. Med. 159, 964-969 51 Hersey, P., Bindon, C. and Czernieck, M. et al. (1983)J. Immunol. 131, 2837-2842 52 Fujiwara, H., Toossi, Z. and EUner, J. J. (1984) Clin. Res. 32,346A 53 Siegel, J. P., Djeu, J. Y. and Stocks, N. I. et aL (1984) Clin. Res. 32, 358A 54 Smith, P. D., Ohura, K. and Masur, H. et aL Glin. Res. 32, 358A 55 Cianeiolo, G.J., Palker, T. and Kipnis, R. et aL (1984)J. Leukocyte Biol. (in press) 56 Palker, T. J., Bolognesi, D. P. and Haynes, B. F. in Current Topics in Immunology (Vogt, P., ed.) (in press) 57 Liotta, L., Tryggvason, K., Garbisa, S., Hart, I., Foltz, C. M. and Shafie, S. (1980) Nature (London) 284, 67-68
The gene for staphylococcal protein A Mathias Uhl6nl'2, M a r t i n Lindberg 2 a n d L e n n a r t Philip son 2'3 Protein A from Staphylococcus aureus has become an important tool in immunology and molecular biology due to its specific binding to the constant region of immunoglobulins (Igs) from most mammalian species 1. Many qualitative and quantitative techniques have been developed which take advantage of this 'pseudo-immune' reaction 2. In addition, solid state protein A has recently been introduced in medical therapy to decrease the amount of circulating immune complexes in sera 3. In this article ~lathias Uhlkn, Martin Lindberg and Lennart Philipson describe the structure of the protein A molecule and its gene. They also discuss the possibilities for fusing the protein A gene to other genes. The gene for protein A has recently been cloned ~'5 allowing studies of the protein and its gene by refined molecular methods. The mechanisms of the specific binding of protein A to different subclasses oflgG as well as the covalent binding of the protein to the peptidog|ycan of the staphylococcal host ceils are problems that can now be approached. By applying site-specific mutagenesis, resulting in predicted amino acid changes in the protein A molecule, the various functions of the protein can be elucidated. Structure and function of the protein
Protein A of S. aureus strain Cowan I is covalently /inked to the peptidoglycan of the cell wall 6'7. Approximately 80 000 binding sites for IgG are present per JDepartment of Biochemistry and Biotechnology. Royal Institute of Technology, S- 100 44 Stockholm, Sweden. 2Department of Microbiology, University of Uppsala, Biomedical Center, Box 581, S-751 23 Uppsala, Sweden. 3European Molecular Biology Laboratory (EMBL), Postfach 1022.09, D-6900 Heidelberg 1, FRG. © 1984,ElsevierSciencePublishersB.V.. Arnsterd~n 0167- 4919/84/$02.00
bacterial cell, uniformly distributed over the surface 8. The interaction between protein A and human IgG shows subclass restriction 9 with strong binding to subclasses IgG1, IgG2 and IgG 4. Some Ig-binding proteins from streptococci seem, however, to lack the subclass restriction 2. Structural studies suggest that each protein A molecule is capable of binding two molecules ofIgG 6a°'~1.Protein A is therefore considered to be functionally bivalent. In contrast, fragmentation of the protein A molecule by trypsin digestion, followed by purification and partial amino acid sequence analysis of the fragments, revealed a tetrameric repeat of the IgG binding part of the molecule (Refs. i0, 12, 13 and Fig. IA). Protein A can be divided into two structurally and functionally different regions. The N-terminal Ig-binding region with a molecular weight of 27 000 consists of four consecutive, mutually homologous IgG-binding domains, each consisting of approximately 58 amino acid residues. The C-terminal region (region X) has a very extended shape and is covalently linked to the cell wall 12. The amino acid sequences of the Ig-binding unks, named fragments D, A, B and C, have been determined 13, re-
Immunology Today, vol. 5, No. 8, 1984
245 been analysed by X-ray crystallography. The complex contains one Fc fragment for every two fragment B units with an overall molecular weight of 57 200 ~s. Fragment B consists of three parallel helical structures arranged in a triangular array with no apparent intramolecular interaction. There is extensive contact between the two N-terminal helices of fragment B and the CH2 and CH3 domains of the Fc fragment. This interaction is mainly hydrophobic although a polar interaction involving only the CH3 domain obviously exists 11.
lllllIIIl[l]" ] NH 2
x~
B
--FS TaqI[
EI DI
l
[ Blcl
Sau3A Bcl]~ . Siu3A . . . . Hiid . lU
"[
×~
X Bcl I Pst .... /
Sau3A[ i 1
0
EcoiV kb
Fig. 1 A. A model of cell wall bound protein A from S. aureus strain Cowan I with the regions determined by protein sequence analysis.
Arrows indicatepointsof enzymaticcleavageby trypsin(TR). The boxes in regionX~indicatethe octapeptiderepeats. Fig. 1 B. Schematic drawing of the gene coding for protein A from strain 8325-4 with its different regions (S is the signal
S. aureus
peptide). Fig. 1 C. Restriction map of the corresponding DNA structure. vealing a hight degree of structural homology. Sj6dah114 showed that fragments A, B and C are monovatent since single fragments do not agglutinate sensitized sheep erythrocytes, but inhibit the reaction between IgG and cells coated with intact protein A. Purified fragment B complexed with the Fc fragment of h u m a n IgG has also
Structure and evolution of the gene
The gene coding for staphylococcal protein A has been isolated by cloning in Escherichia coli using an immunoassay to detect production of the protein 4. The nucleotide sequence of the structural gene has been determined as well as the 5'- and 3'-flanking sequences ~6,17.The gene has many features in common with other genes from Grampositive bacteria 17. These include a strong ribosomebinding sequence complementary to Bacillus subtilis 16S rRNA and a T T G start codon frequently found in Grampositive bacteria~8. Starting from the initiation codon, an open reading frame comprising 1 527 nucleotides gives a preprotein of 509 amino acids and a predicted molecular weight of 58 70317. The nucleotide sequence reveals an additional region called E (Fig. 1B) similar to the repeat Ig-binding regions D-A-B-C reported by Sj6dah112,13. It has not been possible to determine the N-terminal sequence of cell wall
trypsin
GCT GAT AAC AAA TTC AAC AAA GAA CAA CAA AAT GCT TTC TAT GAA ATC TTA AAT ATG CCT Ala Asp Asn Lys Phe Ash Lys Glu Gin Gin Asn Ala Phe Tyr Glu l l e Leu Asn Met P r o l - -T~ - 20
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AAC TTA AAC GAA GAA CAA CGC AAT GGT TTC ATC CAA AGC CTT AAA GAT GAC CCA AGC CAA Ash Leu Asn Glu Glu Gin Arg As__nnGly Phe l l e Gin Ser Leu Lys Asp ~sp Pro Ser Gin 21
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-
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I AGC GCT AAC GTT TTA GCA GAA GCT AAA AAG CTA AAT GAT TCT CAA GCA CCA AAA~ S e r A l a A s n V a l Leu A l a G l u A l a L y s L y s Leu Asn Asp S e r G l n A l a P r o L y s 41 50 58 E D A B C
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Fig. 2. Comparison of the IgG-binding regions with a consensus sequence. Identicalaminoacid residuesare markedwitha line(--), changedresiduesare writtenout and changedcodons, whichdo not givean amino acidchange(wobblechanges),are marked(x). The two a-helicesessentialfor bindingto IgG, and the 11 residuesthat interactwithIgG~Sare also indicated. Note that regionE starts at residue3 and regionD containsa three residueinsert(Ala-Gln-Gln)betweenresidues2 and 3.
246 bound protein A because of an unknown blocking group 12. In contrast protein A from strains exclusively excreting protein A, such as strain A676 and strains containing plasmid pSPA16 (see below), does not have a blocked N-terminus. Amino acid sequencing has also revealed that region E is present, and the N-terminal amino acid is an alanine residue (U. Hellman, unpublished) as predicted from the nucleotide sequence 17. In Fig. 2 the five homologous regions are aligned and compared to a hypothetical ancestral 'consensus' sequence. Mutations resulting in changed amino acids are shown as well as silent mutations, which do not result in a new amino acid (*). The two a-helices essential for binding to IgG, and the 11 residues that interact with IgG ~5 are also indicated. A comparison of the regions with respect to mutual relationship reveals a pronounced 'homology gradient' along the protein molecule, i.e. the closer the location of two regions, the higher the degree of homology 17. As already pointed out by Sj~dah113, one interpretation of this phenomenon is that the ancestral, structural gene coding for the IgG-binding part of protein A has been duplicated during evolution in a step-wise manner, giving slightly dissimilar nucleotide and amino acid sequences. Fig. 2 shows that codons have changed much faster than the amino acids especially in the two a-helices, which suggests an evolutionary pressure to keep the amino acid sequence preserved. Regions A, B and C are highly conserved with no changes in the two a-helices except for a threonine in position 23 of region C. Region D is slightly, more diverged with three changed amino acid residues in the a-helix regions including the asparagine residue at position 11 (Fig. 2), which has been shown to interact with the IgG molecule i~. In addition, region D contains three additional amino acids (Ala-Gln-Gln) after residue no. 2. Region E is even more dissimilar to the consensus sequence. This region consists of only 56 amino acids instead of 58, and the first six residues are different compared to the other regions. However, preliminary studies indicate that regions E and D as single fragments bind to IgG (unpublished observations) although the strength and specificity of the binding has not yet been determined. Cell-wall binding Cell surface proteins of bacteria are considered to be of great importance for the immune response of the host 19. However, so far only a few examples of proteins associated with the bacterial cell wall of Gram-positive bacteria have been characterized and very little is known about the nature of this interaction. Therefore, we decided to study the structure and function of the C-terminal part of protein A, called region X, which is responsible for cell wall binding. The deduced amino acid sequence reveals that this region consists of two structur"ally different parts 16'17(Fig. 1A), the C-terminal region with a unique sequence coding for 81 amino acids (X~) which is preceded by a repetitive region (Xr) of approximately 300 base pairs. The repetitive part of region Xr consists of 12.5 units with a length of 24 nucleotides coding for eight amino acids each. In Fig. 3 the repeats are aligned and a mutual comparison is performed. Six amino acids (Lys-Pro-Gly-Lys-Glu-Asp) are identical
Immunology Today, vol. 5, No. 8, 1984
throughout the X r region. Two amino acids are changed in a regular pattern between Ash-Ash, Gly-Asn or AsnLys. Although the biological function of this extremely conserved octapeptide is not known, clearly there has been a strong pressure to preserve its amino acid sequence. The repetitive structure resembles the structure of another cell surface protein, the streptococcal M protein 19. This protein on the surface of Streptococcus pyogenes (group A streptococcus) cells enables the bacteria to resist ingestion and killing by phagocytic cells. Based on an ahelical structure of the M protein Ofek et aL 20 postulated an alignment of the polyanionic backbone of the lipoteichoic acid with the positively charged amino acids of the M protein. Although binding studies of protein A of strain Cowan 16 indicate interaction with the peptidoglycan instead of the lipoteichoic acid the similarities with the M protein are striking. One of the repeats of the M protein is a heptapeptide Leu-Lys-Thr-Asn-Glu-Gly with an overall structure similar to the octapeptide in region X. Both sequences contain one glycine, two aspartic acids or amides, one glutamic acid and one lysine. A possible function of the repetitive part is to allow the formation of complexes involving ionic interactions. The 12 repeats of 8 amino acid residues in protein A thus might serve to orient such complexes on the outer surface of the cells of S. aureus. The gene for the streptococcal M protein has recently been cloned 21, which will facilitate comparative studies. To confirm that region X is necessary for ceil wall binding two shuttle vectors, able to replicate both in E. coli and S. aureus were constructed 22. Plasmid pSPA15 contains the complete protein A gene, in contrast with plasmid pSPA16 which contains an in-vitro deleted protein A gene encoding a truncated protein A molecule lacking region X. These two plasmids were introduced into different S. aureus strains and the amount of plasmid-coded protein A in the medium (extracellular), as well as bound to the cell wall, was determined 22. All staphylococcal hosts containing plasmid pSPA15 incorporated the plasmid-encoded protein A into the cell wall. When introduced into the methicillin resistant strain A676, which is the most studied producer of extracellular protein A 23, two types of protein A were expressed which can be distinguished by size using SDS-polyacrylamide gel electrophoresis 22. The smaller protein coded by the chromosomal locus was exclusively extracellular, in contrast with the plasmid-coded protein A which was predominantly bound to the cell wall and could be released by lysostaphin treatment. When plasmid pSPA16, lacking region X of the protein A gene, was introduced into strain A676, only extracellular protein A, corresponding to the chromosomal and plasmid encoded genes, could be detected, clearly demonstrating the crucial role of region X in cell wall binding. When plasmid pSPA15 is introduced into coagulasenegative species of staphylococci, such as S. epidermidis, S. xylosus and S. capitis which normally do not produce protein A, the plasmid-encoded protein A is incorporated into the cell wall. In contrast, B. subtilis cells containing a chromosomal insertion vector similar to pSPA15, produce predominantly extracellular protein A (S. Fahnestock, personal communication). Thus region X contains
247
Immunolo~day, voL&No.~1984 AAA
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.
-
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.
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Fig. 3. Comparison of the repetitive units of region X, with a consensus sequence.
Identical amino acid residues are marked with a line (--), changed residues are written out and changed codons, which do not give an amino acidchange(wobblechanges), are marked (x). a structure which anchors the protein to the cell wall of various staphylococcal species, but this interaction is not functional in 13. subtilis. Possibly this is due to differences in cell wall structure or to proteolysis in B. subtilis giving a truncated protein lacking an important domain for binding.* Regulation of the gene
The production of protein A in different strains of S. aureus varies considerably 22'2~. Strain Cowan I produces large amounts (I00 mg 1-~) and strain SAll3 small amounts (15 mg 1- ~) of cell-wall-bound protein A. Strain A676 secretes large amounts (100 mg 1-~) of protein A into the medium and strain Wood 46, although considered as a non-producer of protein A, produces minute amounts. When the shuttle vectors pSPA15 and pSPA16 were transformed into these different hosts 22, the level of expression of the plasmid-encoded protein A varied in accordance with the level of expression of the chromosomal gene encoding protein A, including strain Wood 46 which gave no detectable protein A 22. As the plasmid protein A gene introduced was identical, the levels of expression of the plasmid encoded genes are clearly controlled in the same way as the chromosomal counterpart. Whether the repression of the gene is on the level of transcription, translation or post-translation is yet unknown, but the fact that derepressed strains can be obtained by mutagenesis indicates a specific chromosomal locus responsible for the repression. Gene fusion vectors
Recombinant DNA techniques provide means to splice together the coding sequences of two or more genes. The gene fusion yields hybrid proteins combining the functions of the two parental gene products. This technique can be used for several purposes. First, if the original *A recendy discovered error in the DNA sequence 17of the X-region implies 15 additional amino-acids. Protein A will thereby terminate with a hydrophobic region of 22 amino acids which may be used to anchor the molecule in the cytoplasmic membrane in addition to the interaction between the X-region and the cell wall discussed above.
protein is foreign to the host, rapid proteolytic degradation may be prevented by adding a protective protein as has been demonstrated for the production of somatostatin 24 and insulin 25. Second, the protein may be easier to purify if a protein 'tail' allows affinity chromatography purification2% Third, the proteins can be localized to different compartments (e.g. periplasm, cell wall or medium) through the specific peptides spliced onto the desired protein 27. Fourth, gene fusions can give hybrid proteins with two enzymatic activities. The protein A gene is well suited for such fusions. Four functionally different parts of the gene can be used (Fig. 4). First, the control signals for transcription and translation (promoter, ribosome binding sequence, transcription termination signal) are strong ~7, which results in a high level of expression from any gene spliced into the region. Second, the part of the gene coding for the signal peptide can be used to export proteins through the cytoplasmic membrane of E. ¢0li, B. subtilis or different staphylococcal species2% This might simplify the purification procedure and decrease proteolytic degradation. Third, the part coding for the IgG-binding domains can be used, resulting in hybrid proteins which can be purified or immobilized using IgG coupled to a solid support. The affmity chromatography technique may give a pure and concentrated product, in high yield, by a one-step procedure. It might be possible to trim down the IgG-binding moiety to only 58 amino acid residues and still have this advantage. The fourth part of the protein A molecule which can be used is the cell wall binding domain, called region X. Proteins fused to this domain may bind to the peptidoglycan of staphylococci thus providing an excel-
Structure of the protein A gene "-' "3
,'
I
.....
[.....
owo
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1 Promoter-expression vector
Secretion vector
3
IgG-binding vector (secretion) ,,q
4
IgG-binding vector (intracellular)
5
Cell wall binding vector
Fig. 4. Possible gene fushion vectors based on the protein A gene.
Boxesshowthe relativepositionsfor signalpeptide (SP). IgG-blnding regions (IgG-B), cell wall binding region (CWB) and the foreign gene(FG) splicedintothe vectors.The arrowrepresents transcriptional and translational initiation signals.
Immunology Today, vol. 5, No. 8, 1984
248
lent method for immobilizing enzymes, receptors or other proteins on the cell wall of these bacteria. Two plasmid vectors, pSPAll and pSPA1228, were constructed to allow fusion of other genes to the region of the protein A gene which encodes its IgG binding capacity. A multilinker (from phage M13mp8), containing multiple restriction sites, was inserted into two different parts of the gene. Fusions using vector pSPA11 lead to hybrid proteins lacking region X, in contrast with pSPA12 in which most of region X is intact. The plasmid pSPA12 can therefore be used to produce a hybrid protein with region X as a 'spacer' between the IgG binding moiety and the second protein. As an example of the practical use of such vectors, the protein A gene was fused to the lacZ gene ofE. coli encoding an enzymatically active carboxy-terminal fragment of fl-galactosidase 28. The two resulting plasmids were introduced into an E. coli mutant lacking the lacZ gene. /3galactosidase activity corresponding to the hybrid protein was found in both cases. The hybrid proteins bound efficiently to solid phase IgG with minimal loss of enzymatic activity. Approximately 40-80% of the activity was retained when bound to IgG coated microtiter plates, and 70-80% was retained when the fused proteins were bound to IgG-Sepharose; contrasting with the native/3galactosidase which does not bind. The elution of the hybrid protein was easily achieved by lowering the pH to 3.0. Unfortunately, many enzymes and other proteins are denaturated at this low pH. The elution of fl-galactosidase was instead achieved by competing with pure protein A, when approximately 90% of the activity was eluted. However, for protein analysis or immunization procedures, simple elution at pH 3.0 would probably be more desirable. Suitable plasmid vectors for the different types of gene fusions, as outlined in Fig. 4, are now being constructed and tested. Any fused gene can potentially be expressed giving a hybrid protein which can be purified by affinity chromatography. This will be an interesting alternative for production and purification of proteins, regardless of
whether the protein will be used for immunization, or for structural studies. [~ References 1 Lindmark, R., Thor~n-Tolling, K. and Sj6quist, J. (1983). J. Immunol. Methods 62, 1 2 Langone, J. J. (1982) in Advancesin Immunology(Dixon, F.J. and Kunhel, H. G., eds.), Vol. 32, pp. 157-252, Academic Press, New York 3 Bansal, S. D., Bansal, B. R., Thomas, H. C., Siegel, P. D., Rhoads, J. E., Cooper, D. R., Terman, D. and Mark, R. (1978) Cancer42, 1 4 L6fdahl, S., Guss, B., Uhl~n, M., Philipson, L. and Lindberg, M. (1983) Proc. Natl Aead. Sd. USA 80, 697 5 Duggleby, (3. I. and Jones, S. A. (1983)NacleicAcids Res. 11, 3065 6 SjSquist, J., Movitz, J., Johansson, I.-B. and Hjelm, H. (1972) Ear. J. Biochem. 30, 190 7 Movitz, J. (1974) Eur. J. Biochem. 48, 131 8 Kronvall, G. (1973) Scand. J. ImmunoL 2, 31 9 Kronvall, G. and Williams, R. (3. (1969).J. ImmunoL 103, 828 10 Hjelm, H., Sj6dahl, J. and Sj6quist, J. (1975) Eur. J. Biochem. 57, 395 11 Langone, J. J. (1978).]. Immunol. Methods 24, 269 12 Sj6dahl, J. (1977) Eur. J. Biochem. 73, 343 13 Sj6dabl, J. (1977) EurJ. Biochem. 78, 471 14 Sj6dahl, J. (1976) FEBS Lett. 67, 62 15 Deisenhofer, J. (1981) Biochemistry 20, 2361 16 Guss, B., Uhl~n, M., Nilsson, B., Lindberg, M., Sj6quist, J. and Sj6dahl, J. (1984) Eur. J. Biochem. 138, 413 17 Uhl6n, M., Cuss, B., Nilsson, B., Gatenbeck, S., Philipson, L. and Lindberg, M. (1984).]. BioL Chem. 259, 1695 18 MeLaughlin, J. R., Murray, (3. L. and Rabinowitz, C. (1981)J. BioL Chera. 256, 11283 19 Beaehey, E. H., Seyer, J. M. and Kang, A. H. (1982) in Seminars in Infectious Diseases, Bacterial Vaccines, Vol. 4 (Robbins, J., Hill, J. and Sedoff, J., eds.), pp. 401-410, Thieme-Stratton, Inc., New York 20 Ofek, I., Simpson, W. A. and Beachey, E. H. (1982).]. BacterioL 149, 426 21 Scott, J. R. and Fishetti, V. A. (1983) Science221,758 22 Uhl6n, M., Cuss, B., Nilsson, B., G6tz, F. and Lindberg, M. (1984) J. Bacteriol. (in press) 23 Kronvall, G., Possett, I. H., Quie, P. and Williams, R. C. (1971) Infect. Immun. 3, 10 24 Itakura, K., Hirose, T., Crea, R., Riggs, A. D., Heynecker, H. L., Bolivar, F. and Boyer, H. W. (1977) Science 198, 1056 25 Goeddel, D. V., Kleid, D. G., Bolivar, F., Heynecker, H. L., Yandura, D. G., (3rea, R., Hirose, T., Kraszewski, A., Itakura, K. and Riggs, A. D. (1978) Pr0c. NatlAcad. Sci. USA 76, 106 26 Germino, J., Gray, J. G., Charbonneau, H., Vanaman, T. and Bastia, D. (1983)Proc. NatlAcad. SCL USA 80, 6848 27 Palva, I., Lehtovaara, P., K~i~iinen, L., Sibakov, M., Cantell, K., Schein, C., Kashiwagi, K. and Weissmann, (3. (1983) Gent 22, 229 28 Uhl~n, M., Nilsson, B., Guss, B., Lindberg, M., Gatenbeck, S. and Philipson, L. (1983) Gene23, 369
244 18 Olsen, R. G., Hoover, E. A. and Schaller, j . P. et al. (1977) Cancer Res. 37, 2082-2085 19 Schaner, J. P., Hoover, E. A. and Olsen, R. G. (1977),]. Natl Cancerlnst. 59, 1441-1450 20 Hebebrand, L. C., Mathes, L. E. and Olsen, R. G. (1977) Cancer Res. 39, 443-447 21 Fowler, A. K., Twardzik, D. R. and Reed, C. D. etal. (1977) CancerRes. 37, 4532-4533 " 22 Mathes, L. E., Olsen, R. G. and Hebebrand, L. C. et aL (1978) Nature (London) 274, 687-689 23 Mathes, L. E., Olsen, R. G. and Hebebrand, L. C. et al. (1979) Cancer Res. 39, 950-955 24 Bolognesi, D. P., Montelaro, R. C. and Frank, H. et aL (1978) Science 199, 183-186 25 Denner, J., Wunderlich, V. and Bierwolf, D. (1980)Acta. BioL Med. Ger. 39, 19-26 26 Cianciolo, G.J., Matthews, T.J., Bolognesi, D, P. and Snyderman, R. (1980)J. Immunol. 124, 2900-2905 27 Cianciolo, G.J., Hunter, J , Silva, J. Haskill,J, S. and Snyderman, R. (1981),]. Clin. Invest. 68, 831-844 28 Stiff, M. I. and Olsen, R. G. (1983)J. Gen. ViroL 64, 957-959 29 Copelan, E. A., Rinehart, J . J . and Lewis, M. et al. (1983)J. Immunol. 131, 2017-2020 30 Wellman, M. L., Kociva, G.J. and Lewis, M. G. et aL (1984) Cancer Res. 44, 1527-1529 31 Zbar, B., Wepsic, H. T. and Rapp, H.J. etaL (1970)J. Natl. Cancerlnct. 44, 701-717 32 Snyderman, R., Altman, L. C., Hausman, M. S. and Mergenhagen, S. E. (1972)J. ImmunoL 108, 857-860 33 Boetcher, D. A. and Leonard, E..]. (1974)J. Natl GancerrL Inst. 52, 1091-1099 34 Hausman, M. S., Brosman, S. and Snyderman, R. et aL (1975).]. Natl Cancer Inst. 55, 1047-1054 35 Snyderman, R. and Stahl, C, (1975) in The Phagocytic Cell in Host Resistance (Bellanti, J. A. and Dayton, D. H., eds),'Raven Press, New York
36 Rubin, R. H., Cosimi, A. B. and Goetzl, E. J. (1976) Clin. Immunol. ImmunopathoL 6, 376-388 37 Snyderman, R., Seigler, H. and Meadows, L. (1977)J. Natl GancerInst. 58, 37-41 38 Snyderman, R., Meadows, L., Holder, W. and Wells, S. (1978)J. Natl Cancer Inst 60, 737-740 39 Snyderman, R. and Pike, M. C, (1976) Science 192, 370-372 40 Norman, S.J. and Sorkin, E. (1976),]. Nail Cancer Inst. 57, 135-140 41 Stevenson, M. M. and Mdtzer, M. S. (1976)J. Natl Cancer Inst. 57, 847-852 42 Nelson, M. and Nelson, D. S. (1978) Immunolog~ 34, 277-290 43 Spitalny, G. and North, R.J. (1977)J. Exp. Med. 145, 1264-1277 44 Pike, M. C. and Snyderman, R. (1976),]. Immunol. 117, 1243-1249 45 Cianeiolo, G. J., Herberman, R. B. and Snyderman, R. (1980),]. Natl Cancer Inst. 65, 829-834 46 Levy, M. H. and Wheelock, E. F. (1975),]. ImrnunoL 114, 962-965 47 Bendinelli, M., Toniolo, A. and Friedman, H. (1976) Ann. NYAcad. Sci. 276,431-441 48 Cianciolo, G.J., Lostrom, M. E., Tam, M. and Snyderman, R. (1983) ,jr. Exp. Meal 158, 885-900 49 Cianeiolo, G. J. and Snyderman, R. (1981)J. Clin. Invest. 67, 60-68 50 Cianeiolo, G.J., Phipps, D. and Snyderman, R. (1984)J. Exp. Med. 159, 964-969 51 Hersey, P., Bindon, C. and Czernieck, M. et al. (1983)J. Immunol. 131, 2837-2842 52 Fujiwara, H., Toossi, Z. and EUner, J. J. (1984) Clin. Res. 32,346A 53 Siegel, J. P., Djeu, J. Y. and Stocks, N. I. et aL (1984) Clin. Res. 32, 358A 54 Smith, P. D., Ohura, K. and Masur, H. et aL Glin. Res. 32, 358A 55 Cianeiolo, G.J., Palker, T. and Kipnis, R. et aL (1984)J. Leukocyte Biol. (in press) 56 Palker, T. J., Bolognesi, D. P. and Haynes, B. F. in Current Topics in Immunology (Vogt, P., ed.) (in press) 57 Liotta, L., Tryggvason, K., Garbisa, S., Hart, I., Foltz, C. M. and Shafie, S. (1980) Nature (London) 284, 67-68
The gene for staphylococcal protein A Mathias Uhl6nl'2, M a r t i n Lindberg 2 a n d L e n n a r t Philip son 2'3 Protein A from Staphylococcus aureus has become an important tool in immunology and molecular biology due to its specific binding to the constant region of immunoglobulins (Igs) from most mammalian species 1. Many qualitative and quantitative techniques have been developed which take advantage of this 'pseudo-immune' reaction 2. In addition, solid state protein A has recently been introduced in medical therapy to decrease the amount of circulating immune complexes in sera 3. In this article ~lathias Uhlkn, Martin Lindberg and Lennart Philipson describe the structure of the protein A molecule and its gene. They also discuss the possibilities for fusing the protein A gene to other genes. The gene for protein A has recently been cloned ~'5 allowing studies of the protein and its gene by refined molecular methods. The mechanisms of the specific binding of protein A to different subclasses oflgG as well as the covalent binding of the protein to the peptidog|ycan of the staphylococcal host ceils are problems that can now be approached. By applying site-specific mutagenesis, resulting in predicted amino acid changes in the protein A molecule, the various functions of the protein can be elucidated. Structure and function of the protein
Protein A of S. aureus strain Cowan I is covalently /inked to the peptidoglycan of the cell wall 6'7. Approximately 80 000 binding sites for IgG are present per JDepartment of Biochemistry and Biotechnology. Royal Institute of Technology, S- 100 44 Stockholm, Sweden. 2Department of Microbiology, University of Uppsala, Biomedical Center, Box 581, S-751 23 Uppsala, Sweden. 3European Molecular Biology Laboratory (EMBL), Postfach 1022.09, D-6900 Heidelberg 1, FRG. © 1984,ElsevierSciencePublishersB.V.. Arnsterd~n 0167- 4919/84/$02.00
bacterial cell, uniformly distributed over the surface 8. The interaction between protein A and human IgG shows subclass restriction 9 with strong binding to subclasses IgG1, IgG2 and IgG 4. Some Ig-binding proteins from streptococci seem, however, to lack the subclass restriction 2. Structural studies suggest that each protein A molecule is capable of binding two molecules ofIgG 6a°'~1.Protein A is therefore considered to be functionally bivalent. In contrast, fragmentation of the protein A molecule by trypsin digestion, followed by purification and partial amino acid sequence analysis of the fragments, revealed a tetrameric repeat of the IgG binding part of the molecule (Refs. i0, 12, 13 and Fig. IA). Protein A can be divided into two structurally and functionally different regions. The N-terminal Ig-binding region with a molecular weight of 27 000 consists of four consecutive, mutually homologous IgG-binding domains, each consisting of approximately 58 amino acid residues. The C-terminal region (region X) has a very extended shape and is covalently linked to the cell wall 12. The amino acid sequences of the Ig-binding unks, named fragments D, A, B and C, have been determined 13, re-
Immunology Today, vol. 5, No. 8, 1984
245 been analysed by X-ray crystallography. The complex contains one Fc fragment for every two fragment B units with an overall molecular weight of 57 200 ~s. Fragment B consists of three parallel helical structures arranged in a triangular array with no apparent intramolecular interaction. There is extensive contact between the two N-terminal helices of fragment B and the CH2 and CH3 domains of the Fc fragment. This interaction is mainly hydrophobic although a polar interaction involving only the CH3 domain obviously exists 11.
lllllIIIl[l]" ] NH 2
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"[
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Fig. 1 A. A model of cell wall bound protein A from S. aureus strain Cowan I with the regions determined by protein sequence analysis.
Arrows indicatepointsof enzymaticcleavageby trypsin(TR). The boxes in regionX~indicatethe octapeptiderepeats. Fig. 1 B. Schematic drawing of the gene coding for protein A from strain 8325-4 with its different regions (S is the signal
S. aureus
peptide). Fig. 1 C. Restriction map of the corresponding DNA structure. vealing a hight degree of structural homology. Sj6dah114 showed that fragments A, B and C are monovatent since single fragments do not agglutinate sensitized sheep erythrocytes, but inhibit the reaction between IgG and cells coated with intact protein A. Purified fragment B complexed with the Fc fragment of h u m a n IgG has also
Structure and evolution of the gene
The gene coding for staphylococcal protein A has been isolated by cloning in Escherichia coli using an immunoassay to detect production of the protein 4. The nucleotide sequence of the structural gene has been determined as well as the 5'- and 3'-flanking sequences ~6,17.The gene has many features in common with other genes from Grampositive bacteria 17. These include a strong ribosomebinding sequence complementary to Bacillus subtilis 16S rRNA and a T T G start codon frequently found in Grampositive bacteria~8. Starting from the initiation codon, an open reading frame comprising 1 527 nucleotides gives a preprotein of 509 amino acids and a predicted molecular weight of 58 70317. The nucleotide sequence reveals an additional region called E (Fig. 1B) similar to the repeat Ig-binding regions D-A-B-C reported by Sj6dah112,13. It has not been possible to determine the N-terminal sequence of cell wall
trypsin
GCT GAT AAC AAA TTC AAC AAA GAA CAA CAA AAT GCT TTC TAT GAA ATC TTA AAT ATG CCT Ala Asp Asn Lys Phe Ash Lys Glu Gin Gin Asn Ala Phe Tyr Glu l l e Leu Asn Met P r o l - -T~ - 20
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AAC TTA AAC GAA GAA CAA CGC AAT GGT TTC ATC CAA AGC CTT AAA GAT GAC CCA AGC CAA Ash Leu Asn Glu Glu Gin Arg As__nnGly Phe l l e Gin Ser Leu Lys Asp ~sp Pro Ser Gin 21
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I AGC GCT AAC GTT TTA GCA GAA GCT AAA AAG CTA AAT GAT TCT CAA GCA CCA AAA~ S e r A l a A s n V a l Leu A l a G l u A l a L y s L y s Leu Asn Asp S e r G l n A l a P r o L y s 41 50 58 E D A B C
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Fig. 2. Comparison of the IgG-binding regions with a consensus sequence. Identicalaminoacid residuesare markedwitha line(--), changedresiduesare writtenout and changedcodons, whichdo not givean amino acidchange(wobblechanges),are marked(x). The two a-helicesessentialfor bindingto IgG, and the 11 residuesthat interactwithIgG~Sare also indicated. Note that regionE starts at residue3 and regionD containsa three residueinsert(Ala-Gln-Gln)betweenresidues2 and 3.
246 bound protein A because of an unknown blocking group 12. In contrast protein A from strains exclusively excreting protein A, such as strain A676 and strains containing plasmid pSPA16 (see below), does not have a blocked N-terminus. Amino acid sequencing has also revealed that region E is present, and the N-terminal amino acid is an alanine residue (U. Hellman, unpublished) as predicted from the nucleotide sequence 17. In Fig. 2 the five homologous regions are aligned and compared to a hypothetical ancestral 'consensus' sequence. Mutations resulting in changed amino acids are shown as well as silent mutations, which do not result in a new amino acid (*). The two a-helices essential for binding to IgG, and the 11 residues that interact with IgG ~5 are also indicated. A comparison of the regions with respect to mutual relationship reveals a pronounced 'homology gradient' along the protein molecule, i.e. the closer the location of two regions, the higher the degree of homology 17. As already pointed out by Sj~dah113, one interpretation of this phenomenon is that the ancestral, structural gene coding for the IgG-binding part of protein A has been duplicated during evolution in a step-wise manner, giving slightly dissimilar nucleotide and amino acid sequences. Fig. 2 shows that codons have changed much faster than the amino acids especially in the two a-helices, which suggests an evolutionary pressure to keep the amino acid sequence preserved. Regions A, B and C are highly conserved with no changes in the two a-helices except for a threonine in position 23 of region C. Region D is slightly, more diverged with three changed amino acid residues in the a-helix regions including the asparagine residue at position 11 (Fig. 2), which has been shown to interact with the IgG molecule i~. In addition, region D contains three additional amino acids (Ala-Gln-Gln) after residue no. 2. Region E is even more dissimilar to the consensus sequence. This region consists of only 56 amino acids instead of 58, and the first six residues are different compared to the other regions. However, preliminary studies indicate that regions E and D as single fragments bind to IgG (unpublished observations) although the strength and specificity of the binding has not yet been determined. Cell-wall binding Cell surface proteins of bacteria are considered to be of great importance for the immune response of the host 19. However, so far only a few examples of proteins associated with the bacterial cell wall of Gram-positive bacteria have been characterized and very little is known about the nature of this interaction. Therefore, we decided to study the structure and function of the C-terminal part of protein A, called region X, which is responsible for cell wall binding. The deduced amino acid sequence reveals that this region consists of two structur"ally different parts 16'17(Fig. 1A), the C-terminal region with a unique sequence coding for 81 amino acids (X~) which is preceded by a repetitive region (Xr) of approximately 300 base pairs. The repetitive part of region Xr consists of 12.5 units with a length of 24 nucleotides coding for eight amino acids each. In Fig. 3 the repeats are aligned and a mutual comparison is performed. Six amino acids (Lys-Pro-Gly-Lys-Glu-Asp) are identical
Immunology Today, vol. 5, No. 8, 1984
throughout the X r region. Two amino acids are changed in a regular pattern between Ash-Ash, Gly-Asn or AsnLys. Although the biological function of this extremely conserved octapeptide is not known, clearly there has been a strong pressure to preserve its amino acid sequence. The repetitive structure resembles the structure of another cell surface protein, the streptococcal M protein 19. This protein on the surface of Streptococcus pyogenes (group A streptococcus) cells enables the bacteria to resist ingestion and killing by phagocytic cells. Based on an ahelical structure of the M protein Ofek et aL 20 postulated an alignment of the polyanionic backbone of the lipoteichoic acid with the positively charged amino acids of the M protein. Although binding studies of protein A of strain Cowan 16 indicate interaction with the peptidoglycan instead of the lipoteichoic acid the similarities with the M protein are striking. One of the repeats of the M protein is a heptapeptide Leu-Lys-Thr-Asn-Glu-Gly with an overall structure similar to the octapeptide in region X. Both sequences contain one glycine, two aspartic acids or amides, one glutamic acid and one lysine. A possible function of the repetitive part is to allow the formation of complexes involving ionic interactions. The 12 repeats of 8 amino acid residues in protein A thus might serve to orient such complexes on the outer surface of the cells of S. aureus. The gene for the streptococcal M protein has recently been cloned 21, which will facilitate comparative studies. To confirm that region X is necessary for ceil wall binding two shuttle vectors, able to replicate both in E. coli and S. aureus were constructed 22. Plasmid pSPA15 contains the complete protein A gene, in contrast with plasmid pSPA16 which contains an in-vitro deleted protein A gene encoding a truncated protein A molecule lacking region X. These two plasmids were introduced into different S. aureus strains and the amount of plasmid-coded protein A in the medium (extracellular), as well as bound to the cell wall, was determined 22. All staphylococcal hosts containing plasmid pSPA15 incorporated the plasmid-encoded protein A into the cell wall. When introduced into the methicillin resistant strain A676, which is the most studied producer of extracellular protein A 23, two types of protein A were expressed which can be distinguished by size using SDS-polyacrylamide gel electrophoresis 22. The smaller protein coded by the chromosomal locus was exclusively extracellular, in contrast with the plasmid-coded protein A which was predominantly bound to the cell wall and could be released by lysostaphin treatment. When plasmid pSPA16, lacking region X of the protein A gene, was introduced into strain A676, only extracellular protein A, corresponding to the chromosomal and plasmid encoded genes, could be detected, clearly demonstrating the crucial role of region X in cell wall binding. When plasmid pSPA15 is introduced into coagulasenegative species of staphylococci, such as S. epidermidis, S. xylosus and S. capitis which normally do not produce protein A, the plasmid-encoded protein A is incorporated into the cell wall. In contrast, B. subtilis cells containing a chromosomal insertion vector similar to pSPA15, produce predominantly extracellular protein A (S. Fahnestock, personal communication). Thus region X contains
247
Immunolo~day, voL&No.~1984 AAA
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Fig. 3. Comparison of the repetitive units of region X, with a consensus sequence.
Identical amino acid residues are marked with a line (--), changed residues are written out and changed codons, which do not give an amino acidchange(wobblechanges), are marked (x). a structure which anchors the protein to the cell wall of various staphylococcal species, but this interaction is not functional in 13. subtilis. Possibly this is due to differences in cell wall structure or to proteolysis in B. subtilis giving a truncated protein lacking an important domain for binding.* Regulation of the gene
The production of protein A in different strains of S. aureus varies considerably 22'2~. Strain Cowan I produces large amounts (I00 mg 1-~) and strain SAll3 small amounts (15 mg 1- ~) of cell-wall-bound protein A. Strain A676 secretes large amounts (100 mg 1-~) of protein A into the medium and strain Wood 46, although considered as a non-producer of protein A, produces minute amounts. When the shuttle vectors pSPA15 and pSPA16 were transformed into these different hosts 22, the level of expression of the plasmid-encoded protein A varied in accordance with the level of expression of the chromosomal gene encoding protein A, including strain Wood 46 which gave no detectable protein A 22. As the plasmid protein A gene introduced was identical, the levels of expression of the plasmid encoded genes are clearly controlled in the same way as the chromosomal counterpart. Whether the repression of the gene is on the level of transcription, translation or post-translation is yet unknown, but the fact that derepressed strains can be obtained by mutagenesis indicates a specific chromosomal locus responsible for the repression. Gene fusion vectors
Recombinant DNA techniques provide means to splice together the coding sequences of two or more genes. The gene fusion yields hybrid proteins combining the functions of the two parental gene products. This technique can be used for several purposes. First, if the original *A recendy discovered error in the DNA sequence 17of the X-region implies 15 additional amino-acids. Protein A will thereby terminate with a hydrophobic region of 22 amino acids which may be used to anchor the molecule in the cytoplasmic membrane in addition to the interaction between the X-region and the cell wall discussed above.
protein is foreign to the host, rapid proteolytic degradation may be prevented by adding a protective protein as has been demonstrated for the production of somatostatin 24 and insulin 25. Second, the protein may be easier to purify if a protein 'tail' allows affinity chromatography purification2% Third, the proteins can be localized to different compartments (e.g. periplasm, cell wall or medium) through the specific peptides spliced onto the desired protein 27. Fourth, gene fusions can give hybrid proteins with two enzymatic activities. The protein A gene is well suited for such fusions. Four functionally different parts of the gene can be used (Fig. 4). First, the control signals for transcription and translation (promoter, ribosome binding sequence, transcription termination signal) are strong ~7, which results in a high level of expression from any gene spliced into the region. Second, the part of the gene coding for the signal peptide can be used to export proteins through the cytoplasmic membrane of E. ¢0li, B. subtilis or different staphylococcal species2% This might simplify the purification procedure and decrease proteolytic degradation. Third, the part coding for the IgG-binding domains can be used, resulting in hybrid proteins which can be purified or immobilized using IgG coupled to a solid support. The affmity chromatography technique may give a pure and concentrated product, in high yield, by a one-step procedure. It might be possible to trim down the IgG-binding moiety to only 58 amino acid residues and still have this advantage. The fourth part of the protein A molecule which can be used is the cell wall binding domain, called region X. Proteins fused to this domain may bind to the peptidoglycan of staphylococci thus providing an excel-
Structure of the protein A gene "-' "3
,'
I
.....
[.....
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1 Promoter-expression vector
Secretion vector
3
IgG-binding vector (secretion) ,,q
4
IgG-binding vector (intracellular)
5
Cell wall binding vector
Fig. 4. Possible gene fushion vectors based on the protein A gene.
Boxesshowthe relativepositionsfor signalpeptide (SP). IgG-blnding regions (IgG-B), cell wall binding region (CWB) and the foreign gene(FG) splicedintothe vectors.The arrowrepresents transcriptional and translational initiation signals.
Immunology Today, vol. 5, No. 8, 1984
248
lent method for immobilizing enzymes, receptors or other proteins on the cell wall of these bacteria. Two plasmid vectors, pSPAll and pSPA1228, were constructed to allow fusion of other genes to the region of the protein A gene which encodes its IgG binding capacity. A multilinker (from phage M13mp8), containing multiple restriction sites, was inserted into two different parts of the gene. Fusions using vector pSPA11 lead to hybrid proteins lacking region X, in contrast with pSPA12 in which most of region X is intact. The plasmid pSPA12 can therefore be used to produce a hybrid protein with region X as a 'spacer' between the IgG binding moiety and the second protein. As an example of the practical use of such vectors, the protein A gene was fused to the lacZ gene ofE. coli encoding an enzymatically active carboxy-terminal fragment of fl-galactosidase 28. The two resulting plasmids were introduced into an E. coli mutant lacking the lacZ gene. /3galactosidase activity corresponding to the hybrid protein was found in both cases. The hybrid proteins bound efficiently to solid phase IgG with minimal loss of enzymatic activity. Approximately 40-80% of the activity was retained when bound to IgG coated microtiter plates, and 70-80% was retained when the fused proteins were bound to IgG-Sepharose; contrasting with the native/3galactosidase which does not bind. The elution of the hybrid protein was easily achieved by lowering the pH to 3.0. Unfortunately, many enzymes and other proteins are denaturated at this low pH. The elution of fl-galactosidase was instead achieved by competing with pure protein A, when approximately 90% of the activity was eluted. However, for protein analysis or immunization procedures, simple elution at pH 3.0 would probably be more desirable. Suitable plasmid vectors for the different types of gene fusions, as outlined in Fig. 4, are now being constructed and tested. Any fused gene can potentially be expressed giving a hybrid protein which can be purified by affinity chromatography. This will be an interesting alternative for production and purification of proteins, regardless of
whether the protein will be used for immunization, or for structural studies. [~ References 1 Lindmark, R., Thor~n-Tolling, K. and Sj6quist, J. (1983). J. Immunol. Methods 62, 1 2 Langone, J. J. (1982) in Advancesin Immunology(Dixon, F.J. and Kunhel, H. G., eds.), Vol. 32, pp. 157-252, Academic Press, New York 3 Bansal, S. D., Bansal, B. R., Thomas, H. C., Siegel, P. D., Rhoads, J. E., Cooper, D. R., Terman, D. and Mark, R. (1978) Cancer42, 1 4 L6fdahl, S., Guss, B., Uhl~n, M., Philipson, L. and Lindberg, M. (1983) Proc. Natl Aead. Sd. USA 80, 697 5 Duggleby, (3. I. and Jones, S. A. (1983)NacleicAcids Res. 11, 3065 6 SjSquist, J., Movitz, J., Johansson, I.-B. and Hjelm, H. (1972) Ear. J. Biochem. 30, 190 7 Movitz, J. (1974) Eur. J. Biochem. 48, 131 8 Kronvall, G. (1973) Scand. J. ImmunoL 2, 31 9 Kronvall, G. and Williams, R. (3. (1969).J. ImmunoL 103, 828 10 Hjelm, H., Sj6dahl, J. and Sj6quist, J. (1975) Eur. J. Biochem. 57, 395 11 Langone, J. J. (1978).]. Immunol. Methods 24, 269 12 Sj6dahl, J. (1977) Eur. J. Biochem. 73, 343 13 Sj6dabl, J. (1977) EurJ. Biochem. 78, 471 14 Sj6dahl, J. (1976) FEBS Lett. 67, 62 15 Deisenhofer, J. (1981) Biochemistry 20, 2361 16 Guss, B., Uhl~n, M., Nilsson, B., Lindberg, M., Sj6quist, J. and Sj6dahl, J. (1984) Eur. J. Biochem. 138, 413 17 Uhl6n, M., Cuss, B., Nilsson, B., Gatenbeck, S., Philipson, L. and Lindberg, M. (1984).]. BioL Chem. 259, 1695 18 MeLaughlin, J. R., Murray, (3. L. and Rabinowitz, C. (1981)J. BioL Chera. 256, 11283 19 Beaehey, E. H., Seyer, J. M. and Kang, A. H. (1982) in Seminars in Infectious Diseases, Bacterial Vaccines, Vol. 4 (Robbins, J., Hill, J. and Sedoff, J., eds.), pp. 401-410, Thieme-Stratton, Inc., New York 20 Ofek, I., Simpson, W. A. and Beachey, E. H. (1982).]. BacterioL 149, 426 21 Scott, J. R. and Fishetti, V. A. (1983) Science221,758 22 Uhl6n, M., Cuss, B., Nilsson, B., G6tz, F. and Lindberg, M. (1984) J. Bacteriol. (in press) 23 Kronvall, G., Possett, I. H., Quie, P. and Williams, R. C. (1971) Infect. Immun. 3, 10 24 Itakura, K., Hirose, T., Crea, R., Riggs, A. D., Heynecker, H. L., Bolivar, F. and Boyer, H. W. (1977) Science 198, 1056 25 Goeddel, D. V., Kleid, D. G., Bolivar, F., Heynecker, H. L., Yandura, D. G., (3rea, R., Hirose, T., Kraszewski, A., Itakura, K. and Riggs, A. D. (1978) Pr0c. NatlAcad. Sci. USA 76, 106 26 Germino, J., Gray, J. G., Charbonneau, H., Vanaman, T. and Bastia, D. (1983)Proc. NatlAcad. SCL USA 80, 6848 27 Palva, I., Lehtovaara, P., K~i~iinen, L., Sibakov, M., Cantell, K., Schein, C., Kashiwagi, K. and Weissmann, (3. (1983) Gent 22, 229 28 Uhl~n, M., Nilsson, B., Guss, B., Lindberg, M., Gatenbeck, S. and Philipson, L. (1983) Gene23, 369
