[go: up one dir, main page]

WO1995007300A1 - Procede et moyen de production d'une proteine se liant a la proteine plasmatique - Google Patents

Procede et moyen de production d'une proteine se liant a la proteine plasmatique Download PDF

Info

Publication number
WO1995007300A1
WO1995007300A1 PCT/SE1994/000825 SE9400825W WO9507300A1 WO 1995007300 A1 WO1995007300 A1 WO 1995007300A1 SE 9400825 W SE9400825 W SE 9400825W WO 9507300 A1 WO9507300 A1 WO 9507300A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
binding
mag
albumin
pbs
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/SE1994/000825
Other languages
English (en)
Inventor
Bengt Guss
Hans Jonsson
Martin Lindberg
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to AU76462/94A priority Critical patent/AU7646294A/en
Publication of WO1995007300A1 publication Critical patent/WO1995007300A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to the field of gene technology and is concerned with recombinant DNA molecules, which contain a nucleotide sequence coding for a protein or polypeptide having a broader serum albumin-binding specificity than earlier reported albumin binding properties of protein G from the group G streptococcal strain G148 or other reported streptococcal albumin-binding proteins.
  • the invention comprises microorganisms containing the aforesaid molecules, and the use thereof in the production of the aforesaid protein or polypeptide.
  • the gene encoding protein G from G148 has been cloned and characterized (Guss et al 1986; Olsson et al 1987) . Sj ⁇ bring et al 1989 reported on the structure and distribution of IgG-binding and albumin-binding domains among protein G genes. Further analysis of the serum albumin binding domains of protein G have been reported by Nygren et al 1988; Falkenberg et al 1992; Nygren 1992. The biotechnological use of the serum albumin binding domains of protein G has been documented in several publications and patent applications (WO 9220805; WO 9101743; EP 333691; EP 327522) .
  • serum albumin binding domains of protein G in biotechnology has some drawbacks since not all albumins from different species bind to this protein (Nygren et al 1988; Nygren et al 1990; Bj ⁇ rck and Akerstr ⁇ m 1990) .
  • serum albumins from cow, goat, rabbit, sheep, chicken and horse bind poorly to the albumin binding part of protein G (Nygren et al 1990) or the class a receptors as described by Wideback et al 1983.
  • the animal species from which these albumins originates are from biotechnological, agricultural and commercial aspects very important. In the vaccin development concerning these animal species it should be of great value to have an albumin binding protein with a broader albumin binding spectrum than reported for protein G.
  • albumin binding protein in the biotechnological field a more versatile albumin binding protein would be of value.
  • albumins are present in high amounts added as specific additives or present in a more complex solution such as fetal calf serum (Lillehoj and Malik 1993) .
  • the presence of the albumins could in some cases be a problem when for instance the cell culture media contain products of interest.
  • the presence of albumins in such a solution could be unfavourable since it could have a negative influence on the purification procedure for the product of interest (Lillehoj and Malik 1989, Schneider, 1989). Therefore a protein which for instance binds specifically to albumins from e.g. cow should be of great interest.
  • albumin could in analogy to what Nygren et al 1988 reported be immobilized and used to remove the undesired albumins in the sample. Furthermore a protein (or a fragment thereof) with a broader albumin-binding spectrum could be used to detect and measure the amount of albumin in a sample. Wideback et al 1983 showed that the albumin
  • SUBSTITUTESHEET receptors expressed by S. dysgalactiae could be of interest.
  • S. dysgalactiae type c
  • streptococci are pathogenic and need complex culture media which involves complications in large-scale cultures. There is thus a need for a new method for producing an albumin-binding protein (or fragments thereof) with a broad binding spectrum.
  • the present invention relates to a recombinant DNA molecule comprising a nucleotide sequence which codes for a protein or polypeptide having a broader albumin-binding activity than reported for the class c receptors and protein G or the albumin- binding protein described by Sj ⁇ bring 1992 or Raeder et al 1991.
  • the natural source of this nucleotide sequence is of course the S. dysgalactiae strain 8215 but with the knowledge of the nucleotide and deduced amino acid sequence presented here the gene or parts of the gene can be isolated or made synthetically.
  • the knowledge of the deduced amino acid sequence for the part of the protein responsible for the albumin-binding activity can be used to produce syntethic polypeptides which retain or inhibit the albumin binding.
  • These polypeptides can be labelled with various componds suchs as enzymes, fluorescence, biotin (or derivatives of), radioactivity, etc and used for e.i. in diagnostic tests such as ELISA- or RIA-techniques.
  • a suitable cloning vehicle or vector for example a plasmid or phage DNA
  • a restriction enzyme whereupon the DNA sequence coding for the desired protein or polypeptide is inserted into the cleavage site to form the recombinant DNA molecule.
  • This general procedure is known per se, and various techniques for cleaving and ligating DNA sequences have been described in the literature (see for instance US 4,237,224; Ausubel et al 1991; Sambrook et al 1989), but to our knowledge these techniques have not been used for the present purpose. If the S. dysgalactiae strain 8215 is used as the source of the desired nucleotide sequence it is possible to isolate said sequence and to introduce it into a suitable vector
  • SUBSTITUTESHEET in manner such as described in the experimental part below or, since the nucleotide sequence is presented here, use a polymerase chain reaction (PCR)-technique to obtain the complete or fragments of the mag gene.
  • PCR polymerase chain reaction
  • Hosts that may be used are, microorganisms (which can be made to produce the protein or active fragments thereof) , which may comprise bacterial hosts such as strains of e. g. Escherichia coli , Bacillus subtilis, Streptococcus sp., Staphylococcus sp., Lactobacillus sp. and furthermore yeasts and other eucaryotic cells in culture.
  • regulatory elements such as promoters and ribosome-binding sequences may be varied in a manner known per se.
  • the protein or active peptide thereof can be produced intra- or extracellularly. To obtain good secretion in various bacterial systems different signal peptides could be used.
  • the protein or fragment thereof could be fused to an affinity handle and /or enzyme. This can be done on both genetic and protein level.
  • the gene or parts of the gene can be modified using e.g. in vitro mutagenesis; or by fusion of other nucleotide sequences that encode polypeptides resulting in a fusion protein with new features.
  • the invention thus comprises recombinant DNA molecules containing a nucleotide sequence which codes for a protein or polypeptide having a broad serum albumin-binding activity.
  • the invention comprises vectors such as e.g. plasmids and phages containing such a nucleotide sequence, and organisms, especially bacteria as e.g. strains of E. coli, B. subtilis and Staphylococcus sp. , into which such a vector has been introduced.
  • a nucleotide sequence may be integrated into the natural gene material of the microorganism.
  • the application furthermore relates to methods for production of a protein or polypeptide having the broad albumin-binding activity of protein MAG or active fragments thereof.
  • a microorganism as set forth above is cultured in a suitable medium whereupon the resultant product is isolated by affinity chromatographic purification with the aid of IgG or
  • SUBSTITUTE SHEET albumin bound to an insoluble carrier or by means of some other separating method, for example ion exchange chromatography.
  • Vectors, especially plasmids, which contain the protein MAG encoding nucleotide sequence or parts thereof may advantageously be provided with a readily cleavable restriction site by means of which a nucleotide sequence that codes for another product can be fused to the protein MAG encoding nucleotide sequence, in order to express a so called fusion protein.
  • the fusion protein may be isolated by a procedure utilizing its capacity of binding to serum albumin and/ or IgG, whereupon the other component of the system may if desired be liberated from the fusion protein.
  • the fusion strategy may also be used to modify, increase or change the albumin -binding activity of protein MAG (or albumin binding part thereof) by fusion of other albumin-binding molecules.
  • Streptococcus dysgalactiae strain 8215 was obtained from The National Veterinary Institute, Uppsala, Sweden.
  • E. coli strain DH5 ⁇ was used as bacterial host for the plasmids to be constructed.
  • E. coli strain P2392 was used in cloning with the lambda vector EMBL3 (Frischauf et al 1983) and as a host for expression of the lambda SD1 encoded plasma protein binding protein, termed protein MAG.
  • SUBSTITUTE SHEET used essentially according to the manufacturers recommendations but the host strain was E. coli strain DH5 ⁇ .
  • E. coli was grown on LB (Luria Bertani broth) agar plates or in LB broth (Sambrook et al 1989) at 37 °C. Ampicillin was in appropriate cases added to the E. coli growth media to a final cone, of 50 ⁇ g/ml. Streptococci were grown at 37 °C on bloodagar- plates (containing 5% final cone, bovine blood) or in Todd-Hewitt broth (obtained from Oxoid, Ltd Basingstoke, Hants., England) supplemented with Yeast Extract (Oxoid) to a final cone, of 5%.
  • PBS 0,05M sodium phosphate pH 7,1, 0,9 % NaCl.
  • PBS-T PBS supplemented with TWEEN 20 to a final cone, of 0,05%.
  • S. dysgalactiae strain 8215 was grown overnight in Todd-Hewitt Broth supplemented with 0.6% yeast extract and 10 mM glycine. The next morning glycine was added to a cone. of 0.67 M and the incubation was continued at 37 °C for additional 2 hours. After harvest the cells were washed three times in a buffer consisting of 50 mM Tris-HCl pH 7.0 + 50 mM EDTA and resuspended to 1/20 of the original culture volume in the same buffer including 25% sucrose. Lysozyme ( Boeringer,Germany) was added to a final cone, of 30 mg/ml and the suspension was incubated with gentle agitation for 2 hours at 37 °C.
  • Lysozyme Boeringer,Germany
  • the cells now converted to protoplasts were then pelleted by centrifugation and resuspended in buffer consisting of 50 mM Tris-HCl. pH 7.0 + 50 mM EDTA including 1% SDS (sodium dodecyl sulphate) and incubated at 65°C for 15 minutes. Cell debris was removed by centrifugation and the viscous supernatant further treated as described for chromosomal DNA preparations (Sambrook et al 1989) .
  • Protein G Sepharose 4FF; IgG Sepharose 6FF; CNBr-activated Sepharose 4B was obtained from Pharmacia LKB Biotechnolo-gy, Uppsala, Sweden.
  • Human serum albumin was obtained from Serva (cat. no.11860).
  • Bovine serum albumin (fraction V, ria grade) was obtained from USB (cat. no.10868)
  • DNA probes were labelled with ⁇ 32 P-ATP by a random-priming method (Multiprime DNA labelling system; Amersham Inc, Amersham, England)
  • Nitrocellulose(nc)-filters (Schleicher&Sch ⁇ ll,Dassel,Germany) were used to bind DNA in hybridization experiments or proteins i dot- blot or Western-blot techniques.
  • a gene library of S. dysgalactiae strain 8215 was produced in a manner analogous to that described by Frischholz et al (1983) . Streptococcal DNA was partially digested with Sau3Al and ligated into .BamHI-cleaved lambda EMBL3 vector arms. The ligated DNA was packaged in vitro into phage particles, which were then allowed to infect E. coli P2392 cells. The resultant phage library was analysed for ⁇ 2 M-, BSA- and IgG-binding activity. The resultant phage library was analysed for this purpose on agar plates, in a soft agar layer, which were incubated overnight at 37 °C.
  • the next day plates having a plaque frequency of 10 3 -10 4 were selected.
  • the plaques from each plate were transferred by replica plating to nc-filters. Transfer was allowed to proceed at room temperature for about 15 minutes.
  • the filters were subsequently removed and soaked, using gentle agitation, for 30 minutes in a PBS-T solution (250ml/10 filter with three changes of the PBS- T solution in order to remove loosely bound material such as cell debris and components from the growth media.
  • the filters were then sorted into three groups where replicas originating from the same agarplate were represented in each group.
  • the respective group of filters were transferred to a petri dish which either contained approximately 10 7 cpm of 125 I-labelled rabbit-IgG- antibodies (specific activity 7 mCi/mg) in PBS-T, approximately 10 7 cpm of 125 I-labelled BSA (specific activity 7 mCi/mg) or approximately 10 7 cpm of 125 I-labelled f ⁇ 2 M (125 MBq/ g) .
  • the respective group of filters was washed separately for 3X 10 minutes in 250 ml PBS-T at room temperatur (this washing procedure is important to reduce background signals and has to be prolonged if necessary) . After washing the filters were dried and autoradiographed for several days.
  • Purfied phage DNA from the 1-ambda SD1 clone was analysed by restriction mapping and a preliminary restriction map was constructed. After HindiII digestion of the lambda SD1 DNA, fragments were cloned into the pGEMUZf (+) previously cleaved with Hindlll. After ligation and transformation into E. coli strain DH5 ⁇ recombinant clones were screened for expression of ⁇ 2 M-,BSA- and/or IgG-binding activity. This was done as follows: clones were grown over night on nc-filters on agar plates.
  • the nc-filters were replica plated to a masterplate whereupon the filters were incubated for 10 minutes in chloroform vapour in order to release the proteins from the bacterial cells.
  • the filters were transferred to petri dishes containing respective 15 I-labelled ⁇ X 2 m, BSA or IgG respectively as in Example 1 above. After incubation for 2 hours at room temperature under gentle agitation the filters were washed in PBS-T, dried and autoradiographed as mentioned in Example 1.
  • SUBSTITUTESHEET labelled DNA (random-priming) probe homologous to the domain encoding the IgG-binding regions of protein G was used to identify the presence of homologous sequences among the recombinant plasmid clones.
  • pSDlOl containing a approximately 0,7 kb insert was chosen for further studies.
  • nt sequences of the Hindlll inserts of pSDIOO and pSDlOl were determined and the nt and deduced amino acid (aa) sequences were compared with the corresponding sequences from earlier published sequences of type III Fc receptors (Fahnestock et al. 1986, Guss et al. 1986, Olsson et al. 1987). This analysis revealed only short stretches of homology within the 862 bp HindHI insert of pSDIOO to the other type III Fc receptor genes while the 693 bp HindHI insert of pSDlOl was highly homologous to the earlier studied receptor genes.
  • the combined size of the directly linked inserts of pSDIOO and pSDlOl containing the whole gene, called mag, is 1555 nt (see sequence list, page 27) .
  • mag There is a potential ATG start codon at nt position 288 preceeded by a nt sequence resembling a ribosome-binding site. Upstream this site there are several potential promoter sequences. Starting at the ATG codon there is an open reading frame of 1239 nt terminating in a TAA stop codon at nt 1527.
  • the gene encodes a 413 aa protein, termed protein MAG, with a calculated molecular mass of approximatively 44 kDa including a putative signal peptide.
  • a possible signal peptidase cleavage site should be after the alanine residue at aa position 34 (sequence list, page 27) . Downstream the signal peptide there is a unique strech of 158 aa. No repeated motifs can be seen in this part of the protein from strain 8215. Further to the C-terminal end the deduced aa sequence starting with -ALK- in position 193-195 shows homology to parts of the albumin-binding domain of protein G, the type III Fc receptor from the group G streptococcal strain G148 (Bj ⁇ rck
  • Example 4 (a) . Localization of binding domains in protein MAG.
  • This construct encodes an IgG-binding domain and also the 50 aa sequence upstream that domain which is partially homologous to the albumin-binding domains of protein G (Bj ⁇ rck et al. 1987, Nygren et al. 1988).
  • the subclone pMAG4 encoding the same 50 aa and 10 aa from the IgG-binding domain, was reactive with albumin but not with IgG or f ⁇ 2 M (Figs. IB and C) .
  • the subclone pMAG2 encoding the unique 158 aa long stretch in the N-terminal part of the
  • SUBSTITUTESHEET protein reacted only with ⁇ 2 M.
  • pMAGl lambda SDl was cleaved with PvuII.
  • the approximately 1.1 kb PvuII fragment representing almost the complete mag gene was purified using preparative agarose gel-electrophoresis and ligated into the vector pMALC2 (the vector had previously been cleaved with Ec ⁇ RI and the sticky ends converted to blunt ends using T4 DNA polymerase.
  • pMAG2 the 870 bp Hindlll fragment from pSDIOO was purified by preparative agarose gel-electophoresis.
  • the purified Hindlll- fragment was cleaved by PvuII and a part of the cleaved material was subsequently ligated into the pMALC2 vector (the vector had earlier been cleaved with EcoRI and the generated sticky ends converted to blunt ends using T4 DNA polymerase. After inactivation of the T4 DNA polymerase the vector was cleaved with Hindlll) .
  • pMAG3 the 670 bp Hindlll fragment from pSDlOl was purified by preparative agarose gel-electrophoresis. The purified fragment was cleaved with PvuII and made blunt end with T4 DNA polymerase.
  • the cleaved material was ligated into pMALC2 (the vector had previously been cleaved with -BcoRI and -Ba- ⁇ -HI and the sticky ends converted to blunt ends using T4 DNA polymerase.
  • pMAG4 the 670 bp Hindlll fragment from pSDlOl was purfied using preparative gel-electrophoresis. The purified fragment was cleaved with Hgal and the sticky ends converted to blunt ends using T4 DNA polymerase and ligated into the pMALC2 vector (the vector had earlier been cleaved with EcoRI and SamHI and converted to blunt ends using T4 DNA polymerase. After ligation and transformation into E.
  • coli DH5 ⁇ the generated clones were screened for binding activities as described above. Clones expressing various binding activities were identified and called pMAGl-4. The presence of the expected inserts in pMAGl-4 were verified by nt sequencing (including nt sequencing over the
  • the lambda SDl phages was allowed to adsorb to cells of 3 individual tubes containing 3 ml E. coli P2392 (from an over night culture grown at 37°C in LB-medium supplemented with maltose final cone. 0,2% and MgCl 2 final cone. 10 mM) at a m.o.i. of -0,2 for 15 minutes at 37°C.
  • the phage/bacteria solutions were transferred to three E-flasks, each containg 500 ml LB-medium prewarmed to 37°C. The E-flasks were shaken vigorously at 37°C until lysis occured.
  • the column was sequentially washed with 30 ml PBS, 60 ml PBS-T and 30 ml dH 2 0.
  • the bound protein material was eluted with 12 ml 1 M HAc pH 2,8.
  • the eluted fraction was lyophilized and the dried material was dissolved in a TE-buffer (10ml Tris/HCl pH 7,5; ImM EDTA).
  • SUBSTITUTESHEET Example 6 Binding of protein MAG to serum albumins of different species.
  • S. dysgalactiae belongs to class c, a class showing a broad binding spectrum.
  • this class should bind efficiently to serum albumins from mouse, rat, cow and goat but poorly to serum albumins from humans and horse.
  • albumin binding spectrum of protein MAG the following experiments were performed: albumins of different species were dissolved in PBS and twofold stepwise diluted in PBS in a microtiterplate. The sample in the first well contained ⁇ 10 ⁇ g of the respective kind of albumin while the last well (no.8) contained -0,08.
  • the samples 50 ul were transferred to a nc-filter (previosly soaked in PBS) using a dot-blot apparatus (BioRad, CA, USA) .
  • a dot-blot apparatus BioRad, CA, USA
  • the dot-blot wells were washed twice with 100 ⁇ l PBS and the filter removed and placed in a PBS-T solution (150 ml) .
  • PBS-T solution 150 ml
  • the filter was transferred to a 150ml PBS-T solution to wash away any unbound protein MAG. After 30 minutes at room temperature using gentle agitation and three changes of PBS-T the filter was transferred to a 25 ml PBS-T solution containing goat- anti-rabbit IgG peroxidase conjugate (BioRad) diluted 1/1000. After incubation at room temperature for 1 hour with gentle agitation the filter was transferred to a PBS-T solution (150 ml) to wash away any unbound IgG. After 30 minutes at room temperature using gentle agitation and three changes of PBS-T solution the filter was finally washed twice for 5 minutes in PBS (150 ml) .
  • BioRad goat- anti-rabbit IgG peroxidase conjugate
  • the filter was transferred to a solution containing a substrate for peroxidase (containing 25 ml PBS+ 6 ml 4-chloro-l-naphtol (otained from Sigma, St. Louis, USA) 3mg/ml in methanol + 20 ul H 2 0 2 (35%).
  • protein MAG binds efficiently to albumins of different species.
  • protein MAG binds efficiently to albumins from both human and horses which is not in agreement with the class c receptor type reported by Wideback et al (1983) .
  • the use of the albumin binding properties of protein MAG has several biotechnological advantages compared to other earlier reported streptococcal albumin binding proteins. This since protein MAG binds efficiently to albumins from different species, which are important from agricultural, veterinary, biotechnological and commercial point of view.
  • Example 7 The use of protein MAG in Western-blot techniques
  • Serum albumins from pig, rat, horse, cow, human, goat, mouse and chicken (-0,5 ⁇ g in PBS of respective albumin per well) were run under non-reducing (native) conditions using 8-25% gradient gel in the Phast-system (Pharmacia, Uppsala, Sweden) . After electrophoresis was completed a nc-filter previously soaked in
  • the gradient-gel was after transfer removed and stained with Coomassie-blue using the PHAST-system.
  • the nc-filter was subsequently removed and placed in a 15 ml PBS- T solution containing -10 ⁇ g purified protein MAG. After 1 hour at room temperature using gentle agitation the nc-filter was removed and washed in a 15ml PBS-T at room temperature using gentle agitation for 30 minutes (with two changes of PBS-T solution) .
  • the filter was removed and placed in 15 ml PBS-T solution containing goat-anti-rabbit IgG (Bio-Rad Laboratories, Richmond, CA, USA.
  • Purified protein MAG was dissolved in PBS (lmg/ml) and serially diluted using PBS. Samples from the respective dilution step were transferred to a nc-filter using a dot-blot apparatus (BioRad) The nc-filter was washed with PBS-T and incubated in a solution of PBS-T containing biotinylated bovine serum albumin dilution 1/500. After 1 hour at room temperature using gentle agitation the filter was removed and washed in PBS-T. After washing the filter was incubated 1 hour at room temperature in PBS-T solution containing a streptavidin-horse radish peroxidase conjugate (Amersham,cat.no.
  • Example 9 The use of protein MAG to affinity purify bovine serum albumin from a complex solution
  • Purified protein MAG was coupled to CNBr-activated Sepharose 4B (Pharmacia LKB Biotechnology, Uppsala Sweden following the suppliers general recommendation described in the handbook called "Affinity Chromatography” principles & methods Pharmacia Fine
  • Protein MAG immobilized on Sepharose 4B was subsequently used to affinity purify bovine serum albumin from a complex sample.
  • the sample used was bovine blood (citrate) obtained from The National Veterinary Institute, Uppsala, Sweden. Prior to applying the blood on a column containing protein MAG- Seharose 4B the sample was treated as follows: 10 ml of blood was centrifugated at 12 OOOg for 2 minutes, the supernatant removed and centrifugated at 12 OOOg for 3 minutes, the supernatant taken care of, and 1 ml of this supernatant was diluted ten times using PBS.
  • the diluted sample was passed over a protein MAG-Sepharose 4B column (-0,5 ml sedimentated gel) in order to affinity purify bovine serum albumin. After the sample had passed the column, the column was washed sequentially with 5 ml PBS, followed by 5 ml PBS-T, followed by 5 ml dH20. The bound material on the protein MAG-column was eluted by lowering the pH using 2,4 ml 0,5 M HAc pH 2,8. The eluted sample was lyophilized and the dried sample resuspended in dH 2 0. The sample was analysed using reducing conditions on a SDS-PAGE using a 8-25% gradient gel.
  • bovine and chicken serum albumin (40 ⁇ g of each) were mixed in 1 ml PBS.
  • the mixed albumin solution was passed over a protein MAG-Sepharose 4B column (-0,5 ml sedimentated gel) .
  • the flow through was passed over the column four times to allow maximal binding. After the last passage the flow through was collected. Additional 0,5 ml PBS was added to the column to wash away any unbound material, and the obtained flow through was pooled to the earlier collected 1 ml flow through.
  • the flow through material was desalted and lyophilized.
  • the protein MAG-column was sequentially washed and eluted as described above. The eluted sample was lyophilized.
  • the eluted sample and the sample corresponding to the flow through were resuspended in dH 2 0 and analysed using reducing conditions on a 8-25% gradient-gel using PHAST-system (Pharmacia) . After elecrophoresis the gel was stained with Coomassie-blue.
  • Purified protein MAG (50 ⁇ g) was dissolved in 1 ml PBS.
  • the protein MAG solution was passed over a column containing 0,5 ml bovine serum albumin Agarose (Sigma) previously washed and equilibrated with PBS. The flow through was collected and passed over the column two additional times.
  • the column was sequentially washed with 5 ml PBS, 5 ml PBS-T, 5 ml dH 2 0.
  • the bound material was eluted with 2 ml 0,5 M HAc pH 2,8.
  • the eluted sample was lyophilized and the dried material resuspended in dH 2 0 and analyzed using SDS-PAGE as described above. The result showed that protein MAG was efficiently bound and eluted from the bovine serum albumin column.
  • Example 11 The use of polymerase chain reaction (PCR) to construct clones expressing various number of the albumin-binding domain of protein MAG.
  • PCR polymerase chain reaction
  • the albumin-binding part of protein MAG was cloned using PCR.
  • the Alb-region (sequence list, page 27) was amplified using two synthetic oligonucleotide primers.
  • the underlined nucleotides correspond to nucleotides 864-882 in. the sequence list.
  • the downstream primer 5 '-CGTCTACTGAAGCTAAAATTTCATCTTTAAG- 3 ' was used.
  • the underlined nucleotides correspond to nucleotides 1013-990 in the complementary strand in the sequence list. Plasmid pSDlOl was used as template.
  • the amplified Alb- region was analysed and purified using agarose-gel electrophoresis.
  • the amplified region was ligated into the plasmid vector pUC19 which had previosly been cleaved with
  • the AccI cleaved vector was purified using agarose-gel electrophoresis in order to omit the DNA fragments coding for the Z domains.
  • transformants were screened for production of serum albumin binding activity. This was done as follows; clones were grown over night on nc-filters on agar plates. Next day the nc-filters were replica plated to a master plate whereupon the filters were incubated for 10 minutes in chloroform vapour in order to release the proteins from the bacterial cells. After washing the filters in large excess of PBS-T the filters were transferred to petri dishes containing horse-radish peroxidase labelled human serum albumin.
  • the eluted samples were lyophilized and the dried material resuspended in dH 2 0 and analyzed using SDS-PAGE as described above.
  • the result showed that pMAGALB(PCR)#3, #11 and #21 expresses extracellular polypeptides corresponding to one, two and three Alb-binding domains, respectively(Fig.2A) .
  • the affinity purified Alb-binding domains retain their albumin-binding activity after SDS-PAGE under reduced conditions the samples were transfered by Western-blot as described in Example 7 to a nc-filter.
  • the filter was incubated with horse ⁇ radish peroxidase conjugated human serum albumin for one hour (room temperature under gentle agitation) .
  • Streptococcal proteinG expressed by streptococci or by
  • Escherichia coli has separate binding sites for human albumin and IgG. Mol. Immunol. 24, 1113-1122. Bj ⁇ rck, L. and Akerstr ⁇ m,B. (1990) Streptococcal protein G. In
  • FIG. 1 Schematic presentation of protein products coded by the expression clones pMAG 1-4 derived from S. dysgalactiae strain 8215. Restriction sites used in the construction work are indicated. The two upper lines represent inserts of streptococcal DNA in pSDIOO and pSDlOl. The upper bar represents protein MAG. (The Mai E portion is not drawn to scale) .
  • FIG. 1 SDS-PAGE of HSA-Sepharose affinity purified albumin binding domains expressed by various E. coli clones. Lanes 1-3 corresponds to samples purified from pMAGALB(PCR). #3, #11 and #21, respectively.
  • Sequence list Nucleotide sequence of the mag gene from S. dysgalactiae strain 8215 and the deduced aa sequence. Underlined, the putative transcription initiation signals. The ribosome binding site is double underlined. The start of the signal sequence (S) , ⁇ 2 M-, Albumin- (Alb) and IgG-binding domains, respectively, are indicated as well as the cell wall binding (W) and membrane spanning (M) regions. In the C-terminal the LPXTGX motif is underlined. Methods: The nucleotide sequence was determined for both DNA strands by the dideoxy chain-termination method of Sanger et al. (1977) .
  • Sequence list Nucleotide sequence of the mag gene from S. dysgalactiae strain 8215 and the deduced aa sequence. Underlined, the putative transcription initiation signals. The ribosome binding site is double underlined. The start of the signal sequence (S) , ⁇ 2 M-, Albumin- (Alb) and IgG-binding domains, respectively, are indicated as well as the cell wall binding (W) and membrane spanning (M) regions. In the C-terminal the LPXTGX motif is underlined. Methods: The nucleotide sequence was determined for both DNA strands by the dideoxy chain-termination method of Sanger et al. (1977) .

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Procédé de production d'une protéine présentant l'activité de liaison de l'albumine sérique de la protéine MAG, molécule d'ADN de recombinaison codant pour ladite protéine (ou des fragments de ladite protéine), et micro-organismes contenant cette molécule d'ADN de recombinaison.
PCT/SE1994/000825 1993-09-06 1994-09-06 Procede et moyen de production d'une proteine se liant a la proteine plasmatique Ceased WO1995007300A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU76462/94A AU7646294A (en) 1993-09-06 1994-09-06 Methods and means for producing a plasmaprotein-binding protein

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9302856-1 1993-09-06
SE9302856A SE9302856D0 (sv) 1993-09-06 1993-09-06 Method and means for producing a plasmapeotein-binding protein

Publications (1)

Publication Number Publication Date
WO1995007300A1 true WO1995007300A1 (fr) 1995-03-16

Family

ID=20390978

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE1994/000825 Ceased WO1995007300A1 (fr) 1993-09-06 1994-09-06 Procede et moyen de production d'une proteine se liant a la proteine plasmatique

Country Status (3)

Country Link
AU (1) AU7646294A (fr)
SE (1) SE9302856D0 (fr)
WO (1) WO1995007300A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6100388A (en) * 1998-03-16 2000-08-08 Biogaia Biologies Ab Lactobacilli harboring aggregation gene as a vaccine delivery vehicle

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, File 155, MEDLINE, Dialog Accession No. 06384445, Medline Accession No. 88029445, OLSSON A. et al., "Structure and Evolution of the Repetitive Gene Encoding Streptococcal Protein G"; & EUR. J. BIOCHEM. (GERMANY, WEST) 15 October 1987, 168(2), P319-24.. *
DIALOG INFORMATION SERVICES, File 155, MEDLINE, Dialog Accession No. 08944307, Medline Accession No. 94259307, JONSSON H. et al., "MAG, a Novel Plasma Protein Receptor from Streptococcus Dys-galactiae"; & GENE (NETHERLANDS) 27 May 1994, 143(1), p85-9. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6100388A (en) * 1998-03-16 2000-08-08 Biogaia Biologies Ab Lactobacilli harboring aggregation gene as a vaccine delivery vehicle

Also Published As

Publication number Publication date
AU7646294A (en) 1995-03-27
SE9302856D0 (sv) 1993-09-06

Similar Documents

Publication Publication Date Title
Guss et al. Structure of the IgG‐binding regions of streptococcal protein G.
Jonsson et al. A protein G-related cell surface protein in Streptococcus zooepidemicus
US4977247A (en) Immobilized protein G variants and the use thereof
Rosenow et al. Contribution of novel choline‐binding proteins to adherence, colonization and immunogenicity of Streptococcus pneumoniae
EP0584167B1 (fr) Adn recombine codant pour un peptide signal, polypeptide d'interaction et sequence d'ancrage de membrane
Salamitou et al. Recognition specificity of the duplicated segments present in Clostridium thermocellum endoglucanase CelD and in the cellulosome-integrating protein CipA
AU659139B2 (en) Hybrid polypeptide
Sjöbring Isolation and molecular characterization of a novel albumin-binding protein from group G streptococci
Wanda et al. Purification and characterization of Streptococcus sobrinus dextranase produced in recombinant Escherichia coli and sequence analysis of the dextranase gene
EP0848756A1 (fr) Materiels et procedes relatifs a la fixation et a la presentation de substances sur des surfaces cellulaires
US5180810A (en) Protein h capable of binding to igg
EP0922056B1 (fr) Nouvelle proteine de fixation de fibrinogene tirant son origine du staphylocoque coagulase-negatif
WO1992017588A2 (fr) PROCEDE DE PRODUCTION D'UNE PROTEINE DE LIAISON DE IgA DERIVEE DU GROUPE DES STREPTOCOQUES B
CA2071863C (fr) Vaccin recombinant contre la pleuropneumonie porcine
CN111518174A (zh) 优化的非洲猪瘟CD2v蛋白及其高效表达方法和应用
EP0724596B1 (fr) Procede et moyens de production de proteines se liant a l'inhibiteur de la proteinase plasmatique
EP0293391A1 (fr) Genes streptococcyques clones codant la proteine g et leur utilisation pour construire des micro-organismes recombinants produisant la proteine g
Wang et al. Cloning of the J gene of bacteriophage lambda, expression and solubilization of the J protein: first in vitro studies on the interactions between J and LamB, its cell surface receptor
WO1995007300A1 (fr) Procede et moyen de production d'une proteine se liant a la proteine plasmatique
RU2715672C2 (ru) Рекомбинантные ДНК pG4223 и плазмидная ДНК pQE 30-pG4223, штамм Escherichia coli M 15-G4223, обеспечивающие получение полипептида G4223, селективно связывающего IgG, и его применение в аффинной хроматографии для выделения IgG
JP4049408B2 (ja) グラム陽性菌菌体表面へのタンパク質の固定化方法
AU4270193A (en) Protein l and process for its preparation by recombinant dna technology
JPS62201581A (ja) 新規プラスミド、微生物細胞及びヒト免疫グロブリンＧ　Ｆｃ領域蛋白質の製造方法
Retnoningrum Type M12 protein from Streptococcus pyogenes is a multifunctional receptor

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK ES FI GB GE HU JP KE KG KP KR KZ LK LT LU LV MD MG MN MW NL NO NZ PL PT RO RU SD SE SI SK TJ TT UA US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE MW SD AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA