IgG-BINDING PROTEIN

Abstract: The invention concerns IgG binding proteins H' without albumin binding sequences, DNA sequences coding for the proteins, vectors, host cells containing the DNA, a process for preparing the protein, reagent kit and a pharmaceutical composition. The proteins are useful for a more specific assay and purification of human IgG, removal or adsorption of excessive IgG from blood and for diagnosis of autoimmune diseases than protein H.

Description: igG-binding Protein The present application concerns an IgG-binding protein H, freed from its albumin binding sequence, a DNA-sequence coding for the protein, vectors, host cells containing the DNA, a process for preparing the protein, reagent kit and a pharmaceu¬ tical composition. Protein H is an IgG-binding protein isolated from group A streptococci. (P. Akesson, J. Cooney. F. Kiβhimoto and L. Bjδrck. 1990. Protein H - a novel IgG-binding bacterial protein. Molec. Immunol. 27 523-531). The molecule has a unique structure (H. Gomi, T. H02umi, S. Hattori, C. Tagawa, F. Kiβhimoto and L. Bjδrck. 1990. The gene sequence and some properties of protein H - a novel IgG-binding protein. J. Immunol. 144. 4046-4052) and binds preferentially human IgG. Protein A and protein G, two previously described IgG-binding bacterial proteins, show a much broader IgG-binding capacity, including IgG from most mammalian species. In the case of protein G, the molecule also binds albumin (L. BjSrck, W. Kastern, G. Lindahl and K. widebSck. 1987. Streptococcal protein G, expressed by streptococci or by E. coli. has sepa¬ rate binding sites for human albumin and IgG. Molec. Immunol. 24. 1113-1122). In many situations, the higher degree of speci¬ ficity for human IgG, would make protein H a more valuable tool for binding, detection and purification of human IgG anti¬ bodies. For instance, when human monoclonal IgG antibodies are produced, the growth medium for the antibody-producing cells contains fetal calf βerum. If these antibodies were to be puri¬ fied on a column of protein A or protein G Sepharoβe, such a column would alβo bind IgG from the calf βerum. whereaβ protein H would bind only human IgG. Aβ mentioned above protein G, the IgG-binding protein of human group C and G βtreptococci, also show affinity for albumin. Initial experiments demonstrated that radiolabelled protein H did not bind to albumin immobi¬ lized on nitrocellulose filters (Akesson et al. 1990 supra). However, when protein H was bound to Sepharose, the molecule bound both IgG and albumin. The present invention describes how a non-albumin binding protein H, called protein H' , is con- βtructed and produced. Such a modified protein H molecule could become a powerful tool in biotechnology. It is uβeful for a more specific assay and purification of human IgG, removal or adβorption of exceββive IgG from blood and for diagnosis of autoimmune diβeaβeβ than protein H. By comparing the binding characteriβtics and the amino acid sequence of protein H and protein M6 it was indicated that the albumin binding corresponds to the C and D domains of these proteins. The present invention concerns a non-albumin binding protein H which does not contain the C and/or D domains or parts of the D and/or C domains. The protein according to the invention is capable of binding to the Fc fragment of immunoglobulins and has the following bind¬ ing specifiicity: I) It binds strongly to human lgG(lgGi, lgG2, lgG3 and igG4). human igGFc and rabbit IgG; II) It binds weakly to pig IgG; III) It does not bind to igGβ of mouse, rat, bovine animal, βheep, goat and horβe; IV) It doeβ not bind to human igGFab, igA, IgD, igE and igM; V) It doeβ not bind to human βerum albumin. The protein H without albumin binding sequence, according to the invention is a protein with the amino acid sequence of claim 2 from amino acid nu er l to the end of protein H (amino acid 376 inclusive) minus any fragment within part Cl to the end of part D (from amino acid number 159 to the end of protein H) and subfragmentβ and variants thereof that bind to IgG but not to albumin. This can be a protein with the amino acid sequence starting at amino acid 1 in claim 2 to amino acid number 283 inclusive i.e. the protein H βequence minus the laβt part D and βubfragmentβ and variants thereof that do not bind to albumin but to IgG; or a protein with the amino acid βequence of claim 2 from amino acid number l to amino acid number 241 inclusive i.e. the protein H βequence minus the parts C3 and D of the protein; or a protein with the amino acid βequence of claim 2 from amino acid number l to amino acid number 199 inclusive i.e. the protein H. βequence minus parts C2, C3 and D of the protein H; or a protein with the amino acid sequence of claim 2 from amino acid number l to amino acid number 158 inclusive i.e. parts A and B of protein H. Enclosed are also βubfragmentβ or variantβ of the protein specifically disclosed in the present application wherein the original amino acid βequence iβ modified or altered by inser¬ tion, addition, βubβtitution, inverβion or deletion of one or more amino acids are within the βcope of the present invention aβ far aβ they retain the eββential binding specificity as men¬ tioned above. The invention concerns DNA sequences coding for the proteins mentioned above i.e. from base 1868 to base 2342 inclusive of Fig. 8 plus any fragment from base 2342 to base 2996 that do not give the resulting protein the ability to bind to albumin. The gene coding for the protein H' can be isolated from the chromosomal DNA of a protein H-producing group A βtreptococcal βtrain, βuch aβ streptococcus βp. API based on the information in the DNA βequence of the protein H βhown in Fig. 8 or from the plaβmid p26 (see Fig. 4). The isolation of the gene can also be carried out aβ follows: Chromoβomal DNA can be isolated from cells of the protein H-producing βtrain in accordance with known methodβ (Fahneβtock et al, J. Bacteriol. 167. 870(1986). The iβolated chromo- βomal DNA iβ then segmented into fragments of adequate lengths by biochemical means βuch aβ digestion with a βuitable restric¬ tion enzyme or physical means. The resulting fragments are then inserted at a βuitable reβtriction site into a βuitable cloning vector βuch aβ λgtll (Young et al., Proc. Natl. Acad.Sci. USA BO.. 1194 (1983)) or plaβmid vectors such aβ pUClB (Meβsing et al. , Gene 3_3., 103 (1985)). Alternatively the polymerase chain reaction technique may be used to amplify the 5 1 part of the protein H gene, using primers, the region of the protein H gene coding for the signal, A and B or parts of the C and D region can be selec¬ tively amplified and cloned. A vector βuch as pKC30 can be employed. The recombinantβ are then incorporated into βuitable host cells in this case E. coli. From the resulting transformantβ, the cloneβ producing the protein which bihdβ to human IgG or the Fc region of human IgG, but not to albumin, are selected by a known method (Fahneβtock et al.. βupra) . After the proteinβ capable of binding to human IgG or the Fc region of human IgG, but not to albumin, are iβolated from the resulting positive clones according to conventional methods, the binding specificitieβ of the proteinβ are determined to select the clones producing protein H' . After plas id DNA of said clone iβ iβolated by conventional methods, the DNA βequence of the inβert iβ determined by known methodβ (Sanger et al.. Proc. atl. Acad. Sci. USA 74_, 5463 (1977); Choen et al.. DNA 4_, 165 (1985)). The invention alβo relateβ to DNA βequenceβ that hybridize with said identified DNA βequence under conventional conditionβ and that encode a protein diβplaying essentially the same binding propertieβ aβ said protein H. In this context the term "conven- tional conditions" referβ to hybridization conditionβ where stringent hybridization conditionβ are preferred. The expression of genes may be controlled with vectorβ having the neceββary expression control regions in which only the structural gene iβ inserted. For this purpose, the structural gene shown in Fig. 8 can preferably be used keeping in mind, however, that the whole part Cl to D inclusive must not be preβent. The βtructural gene coding for the protein H 1 can be obtained from the DNA βequence of Fig. 8 or synthesized by conventional methodβ baβed on DNA and amino acid βequenceβ given in the preβent specification. Aβ for the expression vectorβ, variouβ hoβt-vector βyβtemβ have already been developed, from which the most βuitable hoβtvector systems can be selected for the expression of the gene of the preβent invention. It has been known that, for each host cell, there iβ a particu¬ larly preferred codon usage for the expression of a given gene, in constructing a gene to be used for a given hoβt-vector system, the codonβ preferred by the host should be used. Suit¬ able βequenceβ for the gene for the protein H' to be uβed in a particular hoβt-vector system can be designed baβed on the amino acid βequence given in Fig. 8 and βyntheβized by conven¬ tional βynthetic methods. The preβent invention further relateβ to a process for produ¬ cing protein H by culturing a host cell transformed with an expression vector into which the gene encoding protein H is inserted. The proceββ comprises steps of I) inserting a gene coding for protein H into a vector; II) introducing the resulting vector into a βuitable host cell; III) culturing the resulting tranβformant cell to produce protein H; and IV) recovering the protein H from the culture. In the firβt βtep. the gene coding for the protein H. which iβ iβolated from the chromosomal DNA of the API βtreptococcal βtrain and preβent in plaβmid p26 or synthesized aβ mentioned above, iβ inβerted into a vector βuitable for a hoβt to be uβed for the expreββion of the protein H. Inβertion of the gene can be carried out by digeβting the vector with a βuitable restric¬ tion enzyme and linking thereto the gene by a conventional method, βuch as ligation. in the second βtep, the resulting recombinantβ plaβmid iβ introduced into hoβt cellβ. The hoβt cellβ may be Eβcherich coli. Bacillus βubtiliβ or saccharomyceβ cereviβiae or other cellβ. The introduction of the expreββion vector into the hoβt cellβ can be performed in a conventional way and recombinantβ expressing IgG binding peptides are then selected. in the third βtep, the reβulting tranβformant cellβ are cultu¬ red in a βuitable medium to produce protein H by the expreββion of the gene. The cultivation can be conducted in a conventional manner. In the fourth βtep, the produced protein H iβ recovered from the culture and purified, which can be conducted by known methodβ. For example, the cellβ are disrupted by known methodβ βuch aβ ultraβonification, enzyme treatment or grinding. The protein H released by the cellβ or βecreted into the medium iβ recovered and purified by conventional methodβ usually used in the field of biochemistry βuch aβ ion-exchange chromatography, gel filtration, affinity chromatography uβing IgG aβ ligand. hydrophobic chromatography or reversed phase chromatography, which may be uβed alone or in βuitable combinationβ. Aβ mentioned above, the protein provided by the preβent inven¬ tion can be uβed for identification or βeparation of human IgG. For theβe purpoβes, the protein may be brought into a reagent kit or pharmaceutical composition by combining or mixing it with suitable reagents, additives or carriers. When used for treating human blood these additives βhould be pharmaceutically acceptable. Aβ carrierβ there can be uβed activa¬ ted with CNBr, polyacrylamide beads and gold particles. The following figures are attached: Figure l: Dot binding to Ml protein (iβolated from culture βupernatantβ) applied to βtripβ of nitrocellulose in different amounts. The strips were probed with HRPO-labelled proteins. Human βerum albumin (HSA) , fibrinogen (Fbg), human polyclonal immunoglobulin G (IgG), a monoclonal human IgG of βubclaββ 4 (lgG4) and Fc fragments of human IgG (igG-Fc) all bound to Ml protein, whereas F(ab') fragments of IgG (IgG-F(ab') ). human polyclonal immunoglobulin M (igM), vitronectin (vn) and fibronectin (Fn) did not bind. Figure 2: Dot binding to M3 protein (extracted from βtrain 4/55 streptococci with C-phage associated lysin) applied to βtripβ of nitrocellulose in different concentrations. The stripe were probed with HRPO-labelled proteins (for explanation of abbreviations βee legend to Fig. 1). The M3 protein waβ found to bind human βerun albumin (HSA) and fibrinogen (Fbg) strongly, and showed weak affinity for human polyclonal igG(lgG) and fibronectin (Fn) . Figure 3: SDS-polyacrylamide electrophoreβiβ (SDS-PAGE) in a 10% gel of 100 μg βampleβ of extracellular Ml protein waβ followed by transfer of the separated protein βampleβ to βtripβ of nitrocellulose, strips were then probed with different HRPO-labelled proteinβ. The two right lanes show (after stain¬ ing with amidoblack) the separation of Ml protein and molecular weight markers on 10% SDS-PAGE. The figure showβ binding of albumin (HSA), fibrinogen (FbG), polyclonal IgG (IgG), monoclonal lgG'β(lgGl - lgG4) and Fc fragmentβ of igG(lgG-Fc) to Ml protein. Figure 4: Restriction maps of lambda EMBL3 clone 7:1 (a) and plaβmid pK 18 clone p26 (b). Clone pl6 iβ the indicated Sβpl fragment cloned into pUClβ. Figure 5: Nucleic acid and protein βequence of the albumin- binding fragment of βtreptococcal M protein type 1 encoded by pl6. Figure 6: plδ encoded fragment of Ml protein and p26 encoded protein H were applied βeparately to βtripβ of nitrocellulose. The βtripβ were then incubated with HRPO-labelled human βerum albumin (HSA), human fibrinogen or polyclonal human IgG (IgG). The figure βhowβ binding of HSA to the fragment of Ml and to protein H. Protein H alβo bindβ IgG. Figure 7: Compariβon of homologouβ regionβ in protein H, a fragment of M protein type l (pl6) and M protein type 6 (M6). The amino acid βequence of encoded pl6 Ml waβ compared to M6 and protein H, and the homology iβ indicated as percentage identical amino acids. The three βequenceβ were aligned to achieve maximal homology. All three peptideβ bind albumin, which mapβ the albumin-binding activity to the C and/or D regionβ. Figure 8: Nucleic acid βequence of the region of p26 involved in encoding the albumin binding portion of M protein type 1 and protein H. The correβponding amino acid βequenceβ are given. Figure 9: Physical organization of the genes for plβMl and protein H. The thin line repreβentβ the known DNA βequenceβ. The open reading frameβ correβponding to pl6Ml and protein H geneβ are indicated on thiβ line. The division of the genes into βubregionβ is indicated. The base coordinates are under the line whereas the figures above the line are the amino acid coordinates. The βequenceβ correβponding to the inβert DNA in P16 and pPH-l are repreβented, reβpectively, by the two hirozontal broken lineβ. Figure 10: Schematic repreβentation of protein H and two βub¬ fragmentβ and the ability of the correβponding polypeptideβ to bind albumin. Amino acid residue numbers are indicated and βo are the βubregionβ of the peptideβ. Figure ll: Schematic representation of the cloned protein H gene, indicated are s, signal βeguence; A and B unique regions; Cl, C2, C3 repeated βequence; D, D region; w, wall spanning region; M membrane spanning region; T, charged C-terminal resi¬ dues. Horizontal arrowβ labelled I, II. Ill, IV and V indicate the poβition of the primerβ. Figure 12: Shows a map of the truncated protein H gene (protein H') in the expreββion vector pKC30. The ribo ome bind¬ ing βite (rbβ) iβ indicated and βeveral important reβtriction sites. The preβent invention will more preciβely be deβcribed by the following examples. But, they are not intended to limit the βcope of the preβent invention. Example 1 isolation of albumin-binding proteins from Streptococcus pyoqeneβ. Strainβ of streptococcus pyogeneβ M type 1 (40/58) and M 3 (4/55) of the βtrain collection of the WHO Streptococcal Refe¬ rence Laboratory, Prague, Czechoslovakia were grown in 5 1 of Todd Hewitt Broth (Difco) by uβing a 7 1 fermentor. The culture waβ inoculated with 500 ml of an over night βtand culture and fermenation waβ performed for 18 hours at 37 C at pH 7.2 and with regulation of the glucose concentration at 0.2%. The cul¬ ture βupernatantβ and the cell pellets were saved βeparately and uβed for purification of albumin-binding proteinβ. 1. Preparation of albumin-binding proteinβ from culture βuper¬ natantβ Proteinβ in the culture βupernatantβ were precipitated with ammonium sulphate at 70% saturation. The precipitate waβ dialy- zed in the preβence of 50 mM phenylmethylβulfonyl fluoride againβt distilled water and finally against 50 mM sodium ace¬ tate, pH 4.5. This crude preparation waβ applied to 200 ml of CM-Sepharoβe Cl 4B equilibrated with 50 mM Na-acetate, pH 4.5, and waβhed with the same buffer. The column waβ then rinsed with 200 mM sodium phosphate buffer, pH 6.0. Fractions βhowing binding activity to fibrinogen and human βerum albumin (HSA) in dot binding to nitrocelluloβe filter were collected and direct¬ ly applied to affinity chromatography on fibrinogen-Sepharoβe (50 ml). The column waβ waβhed with PBS and bound proteinβ were eluted with 2 M KSCN buffer. Fractionβ which were poβitive in dot binding on nitrocelluloβe against fibrinogen and albumin were collected, dialyzed against 50 mM ammonium hydrogen carbo¬ nate and applied to albumin-Sepharoβe (20 ml). The column waβ waβhed and eluted aβ deβcribed above and fractionβ eluted with 2 M KSCN buffer, which showed fibrinogenand albumin-binding were collected and dialyzed against 50 mM ammonium hydrogen carbonate. The albumin- and fibrinogen-binding material was stored in alliquots or lyophized. 2. Preparation of albumin-binding protein from cell pellets obtained with C-phage aββociated lyβin. Streptococcal cell pellets of the two strainβ (about 60 g of bacteria wet weight) were each suspended in 300 ml PBS contain¬ ing 10 mg Penicillin and 1 ml 2-mercaptoethanol. Then 10 ml of phage lysin prepared according to Cohen et al. (Applied Micro- biol. 29, 175-178, 1975) was added and cells incubated for 6 hours at 37 C with stirring. The resulting lyβate was centri- fuged and the pellet washed with 100 ml of PBS. The resulting βupernatantβ were pooled , dialyzed against water containing 50 mM βodium acetate, pH 4.5, and fractionated on CM-Sepharose as deβcribed above. Albumin- and fibrinogen-binding fractionβ were further purified on fibrinogenand albumin-Sepharoβe, alβo aβ -deβcribed above. Example 2 Characterization of the iβolated albumin-binding βtreptococcal proteinβ Streptococcal M proteinβ (for a review on M protein see Fiβchetti 1989. Clin. Microbiol. Rev. 2, 285) are known to bind fibrinogen and the albumin-binding proteins from both strepto¬ coccal strains (M protein type l and type 3) did indeed show affinity for both fibrinogen and albumin (purified first on fibrinogen-Sepharose and then on albumin-Sepharose) . Further analyβiβ of the proteinβ demonβtrated βerological identity with Ml and M3 protein, reβpectively. Thus, it was concluded that the albumin-binding proteinβ of the two strains represented Ml and M3 proteins. On SDS-polyacrylamide electrophoreβiβ (SDS-PAGE) the estimated molecular weight of Ml waβ 49 kDa, whereaβ the M3 protein waβ more βize heterogeneous with several bands around 60 kDa. In Figβ. l and 2, Ml and M3 proteinβ were applied to nitrocellu¬ loβe filters and probed with some human plasma proteinβ label¬ led with horseradish peroxidaβe (HRPO) aβ described by Tijsβen (In: Laboratory techniqueβ in Biochemistry and Molecular Biology. Eds. Burdon and van Knippenberg. Elsevier, Amsterdam-New York-Oxford 1985). Both M proteins bound albumin. HRPO-labelled Ml and M3 proteinβ were alβo both found to react with albumin applied to nitrocelluloβe filterβ. Aβ shown in figure 1, Ml protein alβo βhowed affinity for HRPO-labelled fibrinogen and immunoglobulin G (IgG). In the case of IgG the interaction waβ mediated through the Fc region. M3 protein βhowed a much weaker affinity for IgG but reacted strongly with HRPO-labelled albumin and fibrinogen (figure 2). The binding properties of Ml protein was alβo studied by Western blot analyβiβ. Aβ βhown in Fig. 3, Ml separated by SDS-PAGE and blotted onto nitrocelluloβe filters, reacted with albumin, fibrinogen and IgG. Example 3 Identification and characterization of the albumin-binding part of M proteinβ and protein H. A genomic library of the βtreptococcuβ M type 1 βtrain 40/58 waβ made in the lambda replacement vector λ EMBL3. The DNA waβ partially cleaved with the restriction enzyme S_au3A and ligated, without βize fractionation, to vector DNA cleaved with Bam HI. Recombinantβ expreββing M protein type l (Ml) were detected uβing βpecific anti-Mi βerum. Three reacting clones were selected for further βtudy. Fig. 4 iβ a phyβical map on one of theβe clones, named λ 7:1.A 4.4 kb Xba I fragment of lambda 7:1 was ligated to the plasmid vector pKlB, and the ligation mix uβed to tranβform E.coli TBl. Tranβformantβ har¬ bouring the correct plaβmid were initially βelected uβing plaβ¬ mid βcreening and confirmed by restriction enzyme digest analy¬ βiβ. one βtrain carrying the correct plaβmid, named p26 waβ βelected for further βtudy. p26 (Fig. 4b) waβ βhown to contain the entire gene βequence for protein H an IgG binding protein of streptococcus, and a truncated Ml gene βequence (Akesson et al. 1990 βupra; Go i et al.1990 βupra). pl6 was generated by βubcloning the 1.6 kb Sβp I fragment of p26. This fragment is indicated in Fig. 4b, and iβ upstream of the protein H gene which iβ located on the β aller Ssp I fragment. The entire inβert of pl6 waβ βequenced and within this βequence an open reading frame correβponding to 281 amino acids was identified (Fig. 5). 14 Lyβateβ of E. coli harbouring either p26 or pl6 were applied to igG-Sepharoβe and albumin-Sepharoβe. respectively. Proteinβ bound were eluted with glycine buffer pH2.0 and 0.15 to 0.63 g of IgG or albumin bindning proteinβ were iβolated per 1 of cul¬ ture of the p26 and pl6 clones, respectively. The N-terminal amino acid βequenceβ of p26 encoded protein H and pl6 encoded Ml were determined (10 amino acidβ each) and found to be fully compatible with the gene βequenceβ of protein H (Gomi et aJL.1990 βupra) and the truncated Ml sequence βhown in Fig. 5. Eluted proteinβ were alβo analyzed in binding experiments. Aβ expected protein H (from the p26 lyβate) reacted with HRPO- labelled human IgG. However, alβo HRPO-labelled albumin reacted with thiβ IgG-binding protein (Fig. 6), whereaβ the protein did not react with albumin when it waβ labelled with 125-1 uβing lactoperoxidaβe or chloramin-T, βuggeβting that thiβ labelling procedure deβtroyβ the albumin-binding activity of protein H. The albumin-binding property of p26 encoded protein H waβ further demonstrated when the protein waβ coupled to sepharoβe. Thus, p26 encoded protein H coupled to Sepharoβe bound albumin (and IgG) very effectively. Finally, protein H did not bind fibrinogen (Fig. 6). The fragment of Ml protein expreββed by pl6, alβo βhowed affinity for albumin. Fibrinogen-binding waβ lost in the pl6 encoded Ml fragment aβ compared to entire Ml (figure 6). Alβo IgG-binding seemed to be loβt, but additional experimentβ βhowed that in βolution pl6 Ml could block the binding of entire Ml protein to IgG, suggesting that part of the igG-binding site iβ preβent on the pi6 Ml protein fragment. To summarize: protein H and M proteins (exemplified with M type 1 and 3 proteinβ) all show affinity for albumin and IgG whereas M proteins, but not protein H, also bind fibrinogen. The amino acid βequence of pl6 Ml waβ then compared to the βequenceβ of protein H (Gomi et al. 1990 βupra) and M protein type 6 (Hollingβhead et al. 1986. J. Biol. Chem. 2j5l 1677). Aβ βhown in Fig. 7 there iβ a βtriking homology between the pl6 encoded Ml βequence and the C-terminal βequenceβ of protein H and M6, indicating that the albumin-binding region corresponds to the C and D domains of these proteinβ. Thus, any protein βequence homologouβ to the βequence encoded by pl6 iβ likely to bind albumin. It iβ alβo intereβting to note that Ml and M3 proteinβ bind IgG. Fibrinogen-binding iβ a well-known property of M proteinβ which waβ alβo demonβtrated for the Ml and M3 protein uβed in thiβ βtudy, whereaβ protein H did not bind fibrinogen. CONCLUSIONS: 1. Proteinβ showing homology with the pl6 sequence of Fig. 5 will most probably bind albumin, in the preβent work thiβ was exemplified with the binding of albumin to M proteins l and 3, a fragment of Ml corresponding to the pl6 sequence of Fig. 5, and protein H. 2. M proteinβ type l and 3 were alβo found to bind IgG and at least in the case of Ml thiβ binding waβ mediated through the Fc region. Example 4 Production of a form of protein H which binds IgG but does not bind albumin. However, a form of protein H which no longer had the ability to bind albumin was generated by deletion of the C-terminal half of the gene. As demonβtrated above albumin binding of protein H is located to C and/or D-regions. Using polymerase chain reaction (PCR) the 5' part of protein H gene waβ amplified. The PCR technique iβ an in vitro method in which genomic or cloned target βequenceβ are βpecifically enzy- matically amplified aβ directed by a pair of oligonucleotide primers. (J.F. Williams. 1989. optimization βtrategieβ for the polymeraβe chain reaction. Biotechniqueβ 7_: 762-768) .Thus, the region of the protein H gene coding for the signal and the A and B region can be selectively amplified and cloned. Using primers I (CAAGGAGTAGATAATGACTAG) and II (TTATGTTTCTAATTGTTGTTGTTTTTGGAGTTG), whoβe poβitionβ are indi¬ cated on the map of protein H gene (Fig. 11), thiβ region waβ obtained. Aβ primer II containβ a low G+C content (9/33 bases) and a run of T reβidueβ, it iβ not an ideal primer (according to the criteria defined by Williams, 1989). sacrificing the last 7 amino acid reβidueβ of the tranβlated B region a possib¬ le primer III (TTATTGGAGTTGTTCTTGATAACG) can be used. This has .8/24 G+C pairs and no run of any single residue. Both primerβ II and III have been deβigned to incorporate βequence which results in an infra e tranβlational βtop codon immediately 3' to the amplified fragment to prevent read- through into vector βequenceβ. Primer I containβ the ribosome binding site of the protein H gene. 0.25 ng of p26 plaβmid DNA waβ uβed as the target for the PCR. The primers were added to a final concentration of 0.1 μM by dilution from a 1 μM stock. The reaction buffer contained 250 μM dATP, dCTP, dGTP and dTTP in 10 mM Triβ-HCl pH 8.3, 50 mM KC1, 1.5 mM MgCl 0.001% gelatine. 100 μl of mineral oil waβ layered over the reaction mix. The DNA waβ denatured by hheeaattiinngg ttoo 9988 Cc aanndd tthheenn ccoooolleedd ttoo : 53°c after which 2 unite of Tag polymeraβe enzyme waβ added. 30 cycleβ of the following βtepβ were performed. Step 1: o l mm at 92 C, step 2: l 1 mmiinn aat 53 C, Step 3: 2 min at 72°C. A Cetuβ intellegent heating block, set on mode 4, was uβed to control the temperature changeβ. After completion of the 30 cycleβ the βample was cooled to room temperature A 10 μl aliguot of the reaction mixture waβ elec- trophoresed on a 3% agaroβe gel. This sample contained 500 ng of a DNA fragment of either 487 bp or 465 bp depending on whether primers I and II or I and III respectively were uβed. Aβ there waβ only one DNA product of thiβ reaction the remain¬ ing βample could be uβed directly for further βtepβ without size fractionation. The reaction mix waβ deproteinized by extraction with phenol saturated with 10 mM Triβ-HCl pH 8.0, l mM EDTA. The extracted material waβ treated with cholorform-iβoamylalcohol (mixed in a 24:1 ratio and saturated with water) to remove excess phenol. The DNA was then precipitated by addition of a 1/10 volume of 8 M LiCl and 2.5 volumes of ice-cold ethanol. The recovered DNA waβ dried under vacuum in a Speedy-Vac and reβuβpended in water to a final concentration of 200 ng/μl 400 ng of product waβ ligated to 200 ng of pKC30 (M. Roβenberg, Y.S. Ho and A. Shatzman. 1989. The use of pKC30 and its derivatives for controlled expreββion of geneβ. pjg., 429-444 in R. wu, L. Grossman and K. Moldave (eds.). Reco binant DNA methodology. Academic Presβ new York) which had been previouβly cleaved with Hpal deproteinized and precipitated aβ deβcribed for the PCR product. The molar ratio of inβert to vector waβ approx. 5:1. The ligation was performed using T4 ligaβe (Gibro BRL) and the buffering syβtem βupplied in accordance with the manufacturer's instructions. Samples of the ligation mix corresponding to 25, 50, 75 and 100 ng of vector DNA were transformed into E. coli βtrain C600 cells lysogenized with λ were made competent uβing the RbCl/CaCl method of Kuβhner (1978). This βtrain doeβ not allow expreββion from the PI of pKC30. Colony hybridization experi entβ (Grundβtein and Hogneββ, 1975) were performed on the reβulting transformantβ uβing the generated PCR fragment aβ a probe. 10 colonieβ which appeared to hybridize were βelected and plaβmid DNA prepared from theβe according to the method of Birnboim and Doly (1979). The DNA waβ further purified by treatment with RNaβe, deproteinized and precipitated. Further hybridization βtudieβ and reβtriction enzyme digeβt analyβiβ of the plaβmidβ confirmed the presence of the desired DNA frag¬ ment. The correct orientation of the insert with reβpect to the PI promoter waβ eβtabliβhed for several of the plasmidβ. The preβence of the correct fragment was confirmed by the abi¬ lity of the purified plaβmidβ to hybridize in Southern blotβ to digoxiginin labelled amplified fragment, uβing Boehringer- Mannheim'β non-radio active DNA labelling kit and by digestion of the plaβmidβ with the reβtriction enzymeβ Tag I - Real in a double digeβt. Thiβ produced a characteriβtic 240 bp fragment which alβo hybridized to the labelled PCR product in Southern blotβ. The orientation of the PCR fragment with reβpect to the promo¬ ter waβ confirmed by the preβence of a 1.9 Kb Hind HI-Rβal fragment which iβ replaced by a 2.1 Kb fragment when the cloned fragment iβ in the opposite orientation to the promotor. See Fig 12 which βhowβ a map of the truncated protein H gene (protein H' ) in the expreββion vector pKC30. The riboβome bind¬ ing βite (rbβ) iβ indicated and several important reβtriction sites. One of the plaβmidβ waβ βelected for further βtudieβ and was called pPH465. This plasmid was transformed into E. coli βtrain pop 2136 aβ described above. Simple plaβmid βcreening establi¬ shed that the intact plaβmid had been tranβformed. The βtrain harbouring pPH465 waβ called PH465. strain PH465 waβ grown in liguid culture at 32°C until the A- 00 was 0.6. Expression of protein H' waβ induced by increaβing the culture temperature to 42° for 20 min and growth waβ continued at 37° for 2 hr. The cellβ were collected by centrifugation and protein H' was purified from cell lysateβ and the culture βupernatent aβ out¬ lined below. If primerβ I and II were employed a 487 baβe pair fragment waβ amplified. The correβponding polypeptide waβ 157 amino acid reβidueβ long (Mr 209.93). If primerβ I and III were uβed a 465 baβe pair fragment reβulted with a polypeptide of 150 amino acidβ (Mr 20112) being encoded. The peptideβ did not bind to albumin. The promoter incorporated in pKC30 iβ the lambda PI promotor. A complete description of the construction of pKC30 and itβ uβeβ are given in Rosenberg et al. 1989 supra. Thiβ promoter was found to be 8-10 times more efficient than Plac. Thiβ high activity haβ been βhown to detrimental in βome caβeβ and can lead to plaβmid instability. By choosing a bacterial hoβt which contains an integrated copy of lambda, the expresβion from PI can be repressed completely by lambda encoded cl (repreββor protein) which increaseβ βtability. The protein H' (the trunca¬ ted gene) clone waβ βelected in thiβ background by colony hybridization using digoxiginin labelled amplified fragment aβ probe and plaβmid DNA waβ prepared. This DNA was uβed to trans¬ form a hoβt which iβ lyβogenized with lambda carrying a tempe¬ rature - βenβitive mutation in cl (CI857) where expreββion of the protein H gene fragment from the PI promotor can be regu¬ lated. Cultures of cells harbouring pKC30/protein H' (the truncated gene) were first grown at 32°C so high cell density was reached without expreββion from PI. Expreββion waβ then induced by increasing the temperature to 42 C. Expreββion waβ rapid - which iβ beneficial aβ the protein H fragment iβ lethal and iβ degraded rapidly. Purification of the protein H fragment The E. coli cellβ from a 5 1 culture were lyβed and the reβult- ing lyβate was subjected to affinity chromatography on igG-Sepharoβe. Bound protein was eluted with glycine buffer pH 2.0, and a single protein band with a molecular weight of 22 kDA on SDS-PAGE was obtained, in total the amount of purified protein was 0.3-0.7 mg/1 culture, in western blotβ thiβ mate¬ rial bound radiolabelled human IgG but not human radiolabelled albumin. Moreover, when the protein H peptide waβ radio label¬ led it bound to IgG-Sepharoβe but not to albumin-Sepharoβe. Finally, the radiolabelled peptide did not form complexeβ with albumin in βolution, aβ judged by immunoprecipitation experi¬ ments with goat anti-human albumin antibodies. Taken together, theβe data demonβtrate that the protein H peptide repreβentβ an IgG-binding fragment of the entire protein H molecule devoid of albumin-binding activity.

Claims: C L A I M S 1. A protein produced by a strain belonging to Group A Streptococci and having the following properties: I) capable of binding βtrongly to human IgG (igGi, igG2, igG3 and lgG4), human IgGFc and rabbit IgG; II) capable of binding weakly to pig IgG; III) incapable of binding to igGs of mouse, rat, bovine animal, sheep, goat and horse; IV) incapable of binding to human IgA, IgD, IgE and igM; V) incapable of binding to albumin and βubfragmentβ and variants thereof with the same properties. 2. A protein according to claim l without albumin binding βequence, characterized in that it haβ the amino acid βequence of protein H below from amino acid number 1 to the end of protein H minus any fragment within part Cl from amino acid number 159 to the end of part D and subfragmentβ and variants therefor that do not bind albumin or especially a protein with the amino acid sequence from acid number l to number 158 inclu¬ sive. v- • v MetThrArgGlnGlnT rLysLysAsnTyrSer euArg ys eu ysThrGlyThrAla 20 v v v v SerValAlaValAlaLeuThrValLeuGlyAlaGlyPheAlaAsnGlnThrThrValLys 40 A r- V V V v AlapluGlyAlaLysIleAspTrpGlnGluGluTyrLysLysLeuAspGluAspAsnAla 60 v v v v LysLeuValGluValValGluThrT rSerLeuGluAsnGluLysLeuLysSerGluAsn 80 v v v v GluGluAsnLys ysAsnLeuAspLysLeuSerLysGluAsnGlnGlyLys euGluLys 100 v v v v LeuGluLeuAspTyrLeuLysLysLeuAspHisGluHisLysGluHisGlnLysGluGln 120 B r* v v v v GlnbluGlnGluGluArgGlnLysAsnGlnGluGlnLeuGluArgLysTyrGlnArgGlu 140 C1 v v v r * v ValGluLysArgTyrGlnGluGlnl-euGlnLysGlnGlnGlnLeuGluThrGlu ysGln 160 v v v v IleSerGluAlaSerArgLysSerLeuSerArgAspLeuGluAlaSerArgAlaAlaLys 180 C2 V V V p £ ysAspLeuGluAlaGluHisGlnLysLeuGluAlaGluHisGlnLysLeuLysGluftsp 200 v v v v ysGlnlleSerAspAlaSerArgGlnGlyLeuSerArgAspLeuGluAlaSerArgAla 220 v v v v AlaLysLysGluLeuGluAlaAsnHisGlnLysLeuGluAlaGluHisGlnLysLeuLys 240 C3 r ~ v v v v GluVspLysGlnlleSerAspAlaSerArgGlnGly euSerArgAspLeuGluAlaSer 260 v v v v ArgAlaAlaLysLysGluLeuGluAlaAsnHisGlnLysLeuGluAlaGluAlaLysAla 280 D . J— V V V V LeuLysGlxjplnLeuAlaLysGlnAlaGluGluLeuAla ysLeuArgAlaGlyLysAla 300 v v v v SerAspSerGlnThrProAspThrLysProGlyAsnLysAlaValProGlyLysGlyGln 32 0 v v v v AlaProGlnAlaGlyThrLysProAsnGlnAsnLysAlaProMetLysGluThrLysArg 340 . v GlnLeuProSerThrGlyGluThrAlaAsnProPhePheThrAlaAlaAlaLeuThrVal 360 v v v v MetAlaThrAlaGiyValAlaAlaValValLysArgLysGluGluAsn 376 SUBSTITUTE SHEET 3. A DNA sequence coding for the protein according to any of the Claims l or 2, aβ it can be deduced from the βeguence below. ATGACTAGACAACAAACCAAGAAAAATTATTCACTACGGAAACTAAAAACCGGTACGGCT 60 v v v v v v TCAGTAGCCGTTGCTTTGACCGTTTTGGGCGCAGGTTTTGCAAACCAAACAACAGTTAAG 120 v v v v v v GCGGAAGGGGCTAAAATTGATTGGCAAGAAGAGTATAAAAAGT AGACGAAGATAATGCT 1 8 ° v v v v v v AAACTTGTTGAGGTTGTTGAAACCACAAGTTTGGAAAACGAAAAACTCAAGAGTGAGAAT 240 V V V V V V GAGGAGAATAAGAAAAATTTAGACAAACT AGCAAAGAAAATCAAGGAAAGCTCGAAAAA 300 V V V V V V TTGGAGCTTGACTATCTCAAAAAATTAGATCACGAGCACAAAGAGCACCAAAAAGAACAA 360 v v v v v v CAAGAACAAGAAGAGCGACAAAAAAATCAAGAACAATTAGAACGTAAATACCAACGAGAA 420 v v v v v v GTAGAAAAACGTTATCAAGAACAAC CCAAAAACAACAACAA'ΠΆGAAACAGAAAAGCAA 8 o v v v v V v ATCTCAGAAGCTAGTCGTAAGAGCCTAAGCCGTGACCTTGAAGCGTCTCGTGCAGCTAAA 540 V V V V V V AAAGACCTTGAAGCTGAGCACCAAAAACTTGAAGCTGAGCACCAAAAACTTAAAGAAGAC 600 v v v v V v AAACAAATCTCAGACGCAAGTCGTCAAGGCC AAGCCGTGACC TGAAGCGTCTCGTGCA 660 V V V V V V GCTAAAAAAGAGCTTGAAGCAAATCACCAAAAACTTGAAGC GAGCACCAAAAACTTAAA 720 V V V V V V GAAGACAAACAAATCTCAGACGCAAGTCGTCAAGGCCTAAGCCGTGACCTTGAAGCGTCT 780 V V V ' V v v CGTGCAGCTAAAAAAGAGCTTGAAGCAAATCACCAAAAACTTGAAGCAGAAGCAAAAGCA 840 V V V V V V CTCAAAGAACAATTAGCGAAACAAGCTGAAGAACTTGCAAAACTAAGAGCTGGAAAAGCA 900 v v v v v v TCAGAC CACAAACCCCTGATACAAAACCAGGAAACAAAGCTGTTCCAGGTAAAGGTCAA 960 V V V V V V GCACCACAAGCAGGTACAAAACCTAACCAAAACAAAGCACCAATGAAGGAAACTAAGAGA 1020 v v v v v v C AG A C CATCAACAGGTGAAACAGCTAACCCATTCT CACAGCGGCAGCCCTTACTGT ° 8 ° v v v v v AT G GCAACAGCTGGAGTAGCAGCAGT GTAAAACGCAAAGAAGAAAACTAA 1131 4. A DNA sequence hybridizing to a DNA βeguence of Claim 3, under conventional conditions and encoding a protein displaying the same binding properties aβ the proteins of any one of Claims l or 2. 5. A recombinant plaβmid vector containing a DNA βequence of any one of Claimβ 3 or 4. 6. A hoβt cell tranβformed with the recombinant plaβmid of Claim 5. 7. A hoβt cell according to Claim 6, which belongs to the βpecieβ E. coli. 8. A process for producing protein H according to Claims l or 2, comprising cultivating a host cell according to Claim 6 or 7 under βuitable conditionβ, accumulating the protein H in the culture or lyβing the. cellβ and recovering it therefrom. 9. A reagent kit for binding, βeparation and identification of human immunoglobulin G, characterized in that it comprises a protein according to any one of Claims 1 or 2. 10. A composition comprising a protein according to any of Claims 1 or 2 and optionally additives or carriers. 11. A pharmaceutical composition comprising a protein according to any of claims l or 2 and optionally pharmaceutical accept¬ able additiveβ or carrierβ.

Inventors: AAKESSON PER, BJOERCK LARS, COONEY JAKKI, SCHMIDT KARL-HERMANN, AAKESSON, PER, BJOERCK, LARS, COONEY, JAKKI, SCHMIDT, KARL-HERMANN
Applicants: HIGHTECH RECEPTOR AB
Application year: 1991
Main classification: C07K13
IPC codes: A61K38, A61P37, C07K14, C07K16, C12N1, C12N15, C12P21, C12R1, G01N33