RU2065746C1 - Recombinant hsa-receptor of streptococcus group g - Google Patents

Abstract

FIELD: molecular biology, genetic engineering. SUBSTANCE: recombinant albumin receptor from human streptococcus group G can bind human serum albumin (HSA) and can be used in immunological investigations. HSA-receptor is obtained by cloning gene G encoding this protein from streptococcus 6148 in E. coli cells, protein isolation from cells by their disruption with 2% sodium dodecyl sulfate (SDS) followed by purification of albumin-binding protein by affinity chromatography. HSA receptor is obtained at high yield and product shows homogeneity that provides high specificity and use in medicinal diagnosis. Obtained protein shows high binding affinity constant with human albumin that ensures to bind its with sorbents that can be used for albumin purification. EFFECT: improved method of HSA-receptor preparing.

Description

 The invention relates to medicine and medical biotechnology. Recombinant Albumin Binding Receptor (HSA) type A from the human group G streptococcus. The HSA receptor has the ability to bind human serum albumin. In addition to human albumin, receptors of this type bind mouse, rat, guinea pig and dog serum albumin, but do not bind bovine, goat and rabbit albumin.

 Due to its ability to bind human serum albumin, the HSA receptor can be used in immunological studies. HSA receptor can be used in ELISA diagnostic test systems to determine the level of albumin in any biological fluids of a person with various types of pathology and normal. The HSA receptor can be an effective ligand for affinity chromatography with which highly purified human and some mammalian albumin can be obtained.

 It is known from the literature that many streptococcal strains expressing the G protein bind human serum albumin. It is known that a full-fledged G protein gene encodes a protein having a molecular weight of 65 kD and having both IgG binding and albumin binding activity. The binding sites for IgG and albumin are located separately in different parts of the G protein molecule, IgG binding receptor is located in the C-terminal part; while albumin binding occurs due to N-terminal domains. (1, 2, 3).

 The HSA receptor can be isolated from streptococcus by enzymatic treatment of cell suspension with papain (native G protein), followed by treatment of this suspension with bacteriolytic agent mutanolysin, or by extraction with 0.6 M HCL during boiling. It can also be obtained by genetic engineering (recombinant protein).

 When processing a streptococcus expressing a G protein with mutanolysin, a 14 kD peptide is formed that binds only HSA, but not IgG. The binding of albumin to a full-sized G protein (65 kDa) is much higher than with a 14 kDa peptide (3). Therefore, the mutanolysin method does not allow the isolation of a complete albumin-binding protein.

Isolation of the HSA receptor by extraction with hydrochloric acid from streptococcus is characterized by the heterogeneity of the resulting product. Thus, it is not possible to completely separate the HSA receptor from IgG binding activity (4). In addition, the isolation of the HSA receptor from streptococcus is characterized by the complexity of the process associated with the use of a conditionally pathogenic microbe. In view of this, it is preferable to obtain a recombinant protein. The HSA receptor is known, obtained by the Swedish authors (5) by cloning the HSA receptor gene in a non-pathogenic host. However, the HSA receptor preparation they described is heterogeneous. In this work, to clone the AB region (the region responsible for albumin binding), the plasmid pSPG2 (6) EcoRl was restricted with subsequent treatment with the Klenov fragment, the addition of a synthetic Sall linker and ligation. Upon restriction of Sall and Pstl, a fragment of 640 bp was obtained. which is isolated from agarose and inserted between the same sites in the pEML8 vector. A fragment of the gene encoding the G protein was excised from the obtained plasmid using EcoRl and Hinh III sites and inserted into the pEG plasmid, the construction of which was described by Eliasson (7). As a result, plasmid pB ₁ B ₂ was obtained, which is responsible for albumin binding activity. From a subclone producing HSA protein, a protein was isolated, purified by affinity chromatography, and examined by SDS-PAGE electrophoresis. As a result, a protein with a molecular weight of 30.5 kD was obtained. The albumin binding protein described by the authors was heterogeneous with the degradation of the purified product.

 The reason for the heterogeneity of the obtained preparation was obviously a rather complicated way of its genetic construction, because initially, a 1.5 kV fragment of the G gene of the protein was taken, which provided expression of only IgG binding activity due to the function of two internal additional promoters. In this case, there was no albumin binding activity, and in order to get it, the authors had to apply a complex construction path using the synthetic Sall linker at the first stage.

 The authors did not give the binding constants of the recombinant albumin receptor to human albumin, and studies of the ability of the HSA receptor to bind to other albumin are also not presented, which does not allow us to compile a complete immunochemical characterization of the receptor they obtained.

 The technical result of our invention is to obtain a homogeneous product of albumin binding protein, characterized by a high binding constant with HSA. The achievement of the technical result is due to an increase in the yield of the target product. In our case, the expression of the albumin binding protein is much higher, since the production is ensured by the function of a powerful promoter from the lactose operon E. coli. The resulting product is homogeneous, because transcription is from the E. coli promoter.

 The essence of the invention is to obtain the HSA receptor by cloning the G gene of the protein from streptococcus G148 into E. coli, isolating the protein from E. coli cells by opening them by boiling in 2% SDS and subsequent purification of the albumin binding protein by affinity chromatography.

 1. Cloning of the HSA receptor. A gene bank of streptococcus G148 was constructed in phage EMBL 3a lambda vector. Of the 50,000 recombinant phage clones, a clone was selected that yielded the highest expression of albumin binding activity. To find the minimum DNA fragment containing the G gene of the protein (in which there are domains responsible for albumin binding activity), subcloning in E. coli JM109 was performed. A 1.5 kb fragment was cloned into pUC8.

 A recombinant 3G plasmid was isolated from the clone giving the highest expression. The clone we obtained possessed both IgG and albumin-binding activity.

 In the approach used by the Swedish authors (5), who used the plasmid obtained by Gooss (6), the reading frame of the lactose operon, in which the G gene of the protein was cloned, was disrupted, therefore, the expression of IgG binding activity was provided by the function of two internal promoters, and albumin binding activity was absent.

 To obtain only the albumin-binding protein, we needed to clone a fragment of a gene that colorates the synthesis of a specific receptor site.

 To maintain this reading frame, in our case, cloning was carried out in the pUC8 vector to the region of the Pstl restriction enzyme site. A 1.5 kb fragment of the protein G gene was also restricted with Pstl. At the same time, the normal reading frame of the lactose operon was preserved, and the part of the gene encoding the albumin binding protein was transcribed without violating the reading frame. In this case, a recombinant product with albumin-binding activity with a high level of expression of the albumin receptor was formed. no IgG binding activity was detected. The recombinant clone selected in this case contained plasmid ra1. The molecular weight of the obtained plasmid was large. By restricting this plasmid with two restriction enzymes EcoR I and Hind III, a 0.64 kb fragment was obtained, which encoded a portion of the protein responsible for albumin binding activity. Subcloning was performed to reduce the plasmid pa1. Restriction of the plasmid pa1 by restriction enzymes EcoR I and Hind III resulted in a 0.64 kv fragment that was isolated from an agarose gel and cloned into plasmid pUC 8, which was restricted with the same restriction enzymes. As a result, the plasmid pa7 was obtained, with a gene fragment encoding the region of the protein responsible for albumin binding activity.

 Thus, an essentially active producer strain is obtained, which may be called Ecoli JM109 a7.

 2. Isolation of the HSA receptor. A culture of Ecoli JM109 a7 was grown overnight in LB medium with ampicillin (100 μg / ml) with strong shootling, then the cells were precipitated by centrifugation at 5000 g, washed, suspended in PBS buffer with the addition of 0.003 M phenylmethylsulfonyl fluoride. Cells were opened by boiling in 2% SDS. After centrifugation at 20,000 g for 1 h, the supernatant was dialyzed against PBS overnight.

 3. Affinity chromatography. The dialysate was applied to a column filled with cyanogen bromide 4B associated with HSA, pre-equilibrated with PBS buffer pH 7.0 with 0.02% sodium azide. Unbound protein was removed by washing the column with the same buffer. HSA binding protein was eluted with 0.1 M glycine buffer, pH 2.0 as a single peak. The pH in the eluate was immediately adjusted to pH 7.0 with a 0.5 M Tris HCL solution, pH 8.8. Albumin binding protein was dialyzed and lyophilized. The yield of the target product of HSA protein is up to 10 mg per liter of culture.

 4. Immunochemical characteristics of the preparation of the recombinant HSA receptor. The HSA receptor drug has been tested for its ability to bind to human serum albumin.

 For this, we used two versions of enzyme-linked immunosorbent assay direct and two-layer (sandwich) methods. In the first of these, the test drug was immobilized on the solid phase and the amount of peroxidase labeled albumin bound to it was estimated. In the second method, an unlabeled albumin preparation was adsorbed on the solid phase, HSA receptor molecules were added from the solution, which, in turn, bound albumin labeled with peroxidase from the solution. In all experiments, at least four parallel titration series were used. The results are presented in graphs 1 and 2.

 Figure 1 (Fig. 4) shows the result of a study of the ability of a solid phase immobilized HSA receptor preparation to bind different concentrations of human albumin.

 Graph 2 (Fig. 5) demonstrates the ability of the HSA receptor, which is in a free (not immobilized) state, to bind human albumin. This graph shows the result of binding of the HSA receptor to human IgG. HSA receptor with lgG did not react.

Immunoblotting. An immunoblot method was used to test the ability of the HSA receptor to bind both human albumin and the albumin of various mammals. Various albumin preparations in various dilutions were applied 2 μl on nitrocellulose filters, which were placed for 30 min in a bLotto solution (2 parts 3% milk + 1 part PBS). The filter was then placed for 30 min in a bLotto solution containing peroxidase-labeled HSA receptor. The filter was washed 3 times for 5 min with bLotto solution and 3 times for 5 min with PBS. The washed filter was dried and placed in a solution with paraoxyphenylene at a concentration of 1 mg / ml with the addition of H ₂ O ₂ . The results of the experiment are presented in FIG. 1, which shows that the HSA receptor efficiently binds human, mouse, rat, and guinea pig albumin; weakly binds bovine and equine albumin, and does not bind rabbit and egg albumin. The interaction of the albumin receptor preparation with human LgA, IgM and IgG was also determined by immunoblotting. The data obtained, (Fig. 2) indicate the absence of interaction of the HSA receptor with human immunoglobulins of classes A, M and G.

Determination of the binding constant of the albumin receptor to human albumin. To determine the equilibrium constant of binding of the HSA receptor to human albumin, the Friguet method (8) was used, which is as follows: antigen at various concentrations is incubated in solution with the antibody at a constant concentration of the latter until equilibrium is reached. The concentration of free antibody is then determined using an indirect enzyme-linked immunosorbent assay. In this case, an HSA receptor was used as a binding agent (antibody), and albumin was used as a ligand (antigen). The equilibrium constant between the HSA receptor and human albumin was 2.7 x ^{10 9} M ^-1 .

 Since the binding constant for the recombinant HSA receptor was not previously determined by other authors, the obtained data can be compared with the binding constant of human albumin with a 14 kD albumin receptor isolated from streptococcus mutanolizine, much lower than that calculated by us, although it turned out to be close to two-receptor full-size G protein the binding constant established by us (3). This once again shows that the 14 kD peptide is only a fragment of the HSA receptor and is not responsible for complete binding to albumin.

 Determination of the molecular weight of the HSA receptor in SDS-PAGE. The HSA receptor preparation was analyzed in SDS-PAGE according to the Laemmli method (9). The molecular weight of the obtained HSA receptor was MB 31 kD (Fig. 3). The following were used as molecular weight standards: phosphorylase B with MB 94000; bovine serum albumin with a molecular weight of 67,000; ovalbulin with MW 43000; bovine carbonic anhydrase with an MW of 30,000; trypsin inhibitor with a MW of 20,000 and egg lysozyme with a MW of 14,000.

The stability of the drug HSA receptor. The HSA receptor is readily soluble in water and aqueous solutions, can be easily lyophilized, frozen several times, and is stable when stored in solutions at 10 ^o C. The HSA receptor can be easily labeled with peroxidase, alkaline phosphatase and radioactive iodine, which allows its use in various immunological test systems.

 The nucleotide sequence and the derived amino acid sequence, amino acid designation and amino acid composition, see the end of the description text.

 Determination of the isoelectric point (pI) of the HSA receptor. The isoelectric point is determined by isoelectric focusing. The HSA receptor focuses on Phast gellEF with the following standards: glucose oxidase pI 4.15, trypsin inhibitor pI 4.55, beta-lactoglobulin pI 5.2, bovine carbonic anhydrase B, pI 5.85, human carbonic anhydrase, pI 6.55 . Defined pI of albumin receptor 5.8.

Compared with the known and previously described proteins with the ability to react with albumin, the HSA receptor we offer has a number of significant advantages:
1. High yield of HSA receptor (up to 10 mg per 1. culture). For the HSA preparations described in the literature, the protein yield is not given and this drug is not presented in any of the catalogs of foreign companies. Thus, this drug is absolutely original, which ensures its high competitiveness in the world market.

 2. The homogeneity of the obtained albumin-binding protein ensures its high specificity and makes it possible to use in medical diagnostics of renal and other forms of pathology, where the disease is characterized by a change in the level of albumin in human biological fluids.

 3. The high affinity of the HSA receptor allows you to bind it to the sorbents used in affinity chromatography for the purification of human albumin and the albumin of some mammals or to obtain pure biological preparations in which albumin is present as a ballast protein.

 4. The obtained HSA receptor is essentially completely original, with high specificity, which should allow to replace anti-albumin sera in diagnostic systems for determining albumin in biological fluids, which are obtained in a laborious way, at high cost, and which do not have strict immunological specificity and can produce cross reactions.

 In connection with the above, the HSA receptor we have obtained has the prospect of widespread use in applied immunology, immunochemistry, medical diagnostics, and also for industrial purposes.

SIGNATURES TO FIGURES
Schedule 1 (Fig. 4). The ability of a solid phase immobilized HSA receptor to bind various concentrations of albumin.

 Schedule 2 (Fig. 5). The binding of the HSA receptor to albumin and lgG.

FIG. 1. Immunoblotting. The binding of the HSA receptor to albumin (double dilutions with an initial concentration of 1 mg / ml):
1) egg albumin
2) rabbit
3) horses
4) guinea pig
5) mice
6) person
FIG. 2. Immunoblotting. The binding of the HSA receptor to albumin and human immunoglobulins of classes A, M, G (double dilutions):
1) lgM
2) lgA
3) logG
4) albumin
FIG. 3. SDS-PAGE albumin binding protein purified by affinity chromatography.

1) lysate of the HSA receptor
2) HSA receptor after affinity chromatography (concentration 10 μg)
3) molecular weight marker
4) HSA receptor after affinity chromatography (concentration 25 μg).

Sources
1. Akerstrom B. Brodin T. Reis K. and Bjorck L. 1985, JN. Immunol. 135, 2589-2592.

 2. Bjorck L. Kastern W. Lindahl G. Wideback K. 1987, Mol. Immunol. 24, 1113-1122.

 3. Sjobring U. Falkenberg C. Nielson E. Akerstrom B. Bjork L. 1988, Immunol. 140, 1595-1599.

 4. Wiedeback K. Lund 1987, 92.

 5. Nygren P.-A. Eliasson M. Abrahmsen L. Uhlen M. 1988, J. Molec. Recognition 1, 69-74.

 6. Guss B. Eliasson M. Olsson A. Uhlen M. Frej A-K. Jornvall H. Flock J. Lindberg M. 1986, EMBO J, 5, 1567-1575.

 7. Eliasson M. Olsson A. Palmcronitz E. Wiberg K. Inganas M. Guss B. Lindberg M. Uhlen M. 1988, J. Biol. Chem. 263, 4323-4327.

 8. Friquent B. Chaffotte A.B. 1985, J. Immunol. Meth. 75, 305-319.

 9. Laemmli U.K. 1970, Nature 227, 680-685.

 10. Sanger F. Coulson A.R. Barell B.G. Smith A.J. Roc B.A. 1980, J. Mol. Biol. 143, 161-178. YYY2 YYY4 YYY6

Claims (4)

 1. The HSA protein obtained by cloning the HSA receptor gene from streptococcus C148 in a non-pathogenic strain Ecoli JM 109 transformed with the recombinant plasmid pa 7 containing the nucleotide sequence of the structural part of the albumin receptor gene described in the description, wherein the protein is characterized by the amino acid sequence shown in description, and mol.m. 31 kD. 2. The HSA protein according to claim 1 is characterized by a binding constant for human albumin of 2.7 x 10 ⁹ M ^{- 1} .  3. The HSA protein according to claims 1 and 2 is characterized in that 1 mg of the receptor binds 7 mg of human albumin in solution.  4. The HSA protein according to claims 1 to 3 is characterized by an isoelectric point p 15.8.

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