Definition of IgG- and albumin-binding regions of streptococcal protein G

Protein G, the immunoglobin G-binding surface protein of group C and G streptococci, also binds serum albumin. The albumin-binding site on protein G is distinct from the immunoglobulin G-binding site. By mild acid hydrolysis of the papain-liberated protein G fragment (35 kDa), a 28-kDa fragment was produced which retained full immunoglobulin G-binding activity (determined by Scatchard plotting) but had lost all albumin-binding capacity. A protein G (65 kDa), isolated after cloning and expression of the protein G gene in Escherichia coli, had comparable affinity to immunoglobulin G (5-10 X 10(10)M-1), but much higher affinity to albumin than the 35- and 28-kDa protein G fragments (31, 2.6, and 0 X 10(9)M-1, respectively). The amino-terminal amino acid sequences of the 65-, 35-, and 28-kDa fragments allowed us to exactly locate the three fragments in an overall sequence map of protein G, based on the partial gene sequences published by Guss et al. (Guss, B., Eliasson, M., Olsson, A., Uhlen, M., Frej, A.-K., J?rnvall, H., Flock, J.-I., and Lindberg, M. (1986) EMBO J. 5, 1567-1575) and Fahnestock et al. (Fahnestock, S. R., Alexander, P., Nagle, J., and Filpula, D. (1986) J. Bacteriol. 167, 870-880). In this map could then be deduced the location of three homologous albumin-binding regions and three homologous immunoglobulin G-binding regions.     

Isolation and characterization of a 40-kDa cyclophilin-related protein.

Major and minor isoforms of cyclophilin (CyP-18), a 17.8-kDa protein with peptidyl-prolyl cis-trans isomerase activity, comprise the primary intracellular binding proteins for cyclosporin A. Additional CyP-like proteins with approximate molecular masses of 22 (CyP-22) and 40 kDa (CyP-40) have been recovered from the soluble fraction of calf brain along with CyP-18 by adsorption onto a cyclosporin A affinity column and elution with cyclosporin A. Based on a limited number of peptide sequences from CyP-22, it appears that we have isolated from tissue CyPB, a protein whose sequence was deduced previously from cloned cDNA. The 40-kDa protein was separated from CyP-18 and CyP-22 on a molecular sieving column. Isoelectric focusing of CyP-40 yielded two bands at pI 5.3 and 5.5, in contrast to the basic pI values of CyP-18. Some tryptic peptides from CyP-40 were found to be highly homologous but not identical to bovine CyP-18; others were not significantly homologous to CyP-18 or any other protein in the data base. Unlike the major and minor isoforms of Cyp-18, monospecific polyclonal anti-CyP-18 antibodies did not cross-react with CyP-22 and CyP-40. Likewise, anti-CyP-40 serum minimally cross-reacts with CyP-18 and CyP-22. Cyp-40 possesses peptidyl-prolyl cis-trans isomerase activity which is less sensitive to inhibition by cyclosporin A (IC50 = 300 nM) than is CyP-18 (IC50 = 20 nM).     

Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin

Streptococcal protein G (SpG) is a bacterial cell surface receptor exhibiting affinity to both human immunoglobulin (IgG) and human serum albumin (HSA). Interestingly, the serum albumin and immunoglobulin-binding activities have been shown to reside at functionally and structurally separated receptor domains. The binding domain of the HSA-binding part has been shown to be a 46-residue triple alpha-helical structure, but the binding site to HSA has not yet been determined. Here, we have investigated the precise binding region of this bacterial receptor by protein engineering applying an alanine-scanning procedure followed by binding studies by surface plasmon resonance (SPR). The secondary structure as well as the HSA binding of the resulting albumin-binding domain (ABD) variants were analyzed using circular dichroism (CD) and affinity blotting. The analysis shows that the HSA binding involves residues mainly in the second alpha-helix.     

Practical applications of high-affinity, albumin-binding proteins from a group G streptococcal isolate

Binding proteins that have high affinities for mammalian plasma proteins that are expressed on the surface of bacteria have proven valuable for the purification and detection of several biologically important molecules from human and animal plasma or serum. In this study, we have isolated a high affinity albumin-binding molecule from a group G streptococcal isolate of bovine origin and have demonstrated that the isolated protein can be biotinylated without loss of binding activity and can be used as a tracer for quantification of human serum albumin (HSA). The binding protein can be immobilized and used as a selective capture reagent in a competitive ELISA format using a biotinylated HSA tracer. In this assay format, the sensitivity of detection for 50% inhibition of binding of HSA was less than 1 μg/ml. When attached to the bacterial surface, this binding protein can be used to deplete albumin from human plasma, as analyzed by surface-enhanced laser desorption ionization time of flight mass spectrometry.     

Isolation of a 40-kDa Huntingtin-associated protein

Huntington's disease is caused by an expanded CAG trinucleotide repeat coding for a polyglutamine stretch within the huntingtin protein. Currently, the function of normal huntingtin and the mechanism by which expanded huntingtin causes selective neurotoxicity remain unknown. Clues may come from the identification of huntingtin-associated proteins (HAPs). Here, we show that huntingtin copurifies with a single novel 40-kDa protein termed HAP40. HAP40 is encoded by the open reading frame factor VIII-associated gene A (F8A) located within intron 22 of the factor VIII gene. In transfected cell extracts, HAP40 coimmunoprecipitates with full-length huntingtin but not with an N-terminal huntingtin fragment. Recombinant HAP40 is cytoplasmic in the presence of huntingtin but is actively targeted to the nucleus in the absence of huntingtin. These data indicate that HAP40 is likely to contribute to the function of normal huntingtin and is a candidate for involvement in the aberrant nuclear localization of mutant huntingtin found in degenerating neurons in Huntington's disease.     

Fragment reconstitution of a small protein: disulfide mutant of a short C-terminal fragment derived from streptococcal protein G

Hierarchical studies on the folding of protein G B1 domain have shown that the C-terminal fragment (C16) has a considerable amount of beta-hairpin structure that exchanges between the folded and unfolded states at room temperature, and that the C16 fragment binds noncovalently to an N-terminal fragment (N40) under physiological conditions. Those studies have led us to the hypothesis that the amphipathic beta-hairpin structure of C16 initiates folding of the domain. To obtain a more detailed understanding of the folding mechanism of the domain, we designed a mutant of C16 (SS16ox) with a disulfide bond between residues 41 and 56, and then examined the interaction of the mutant with N40 by surface plasmon resonance (SPR) and by thermal denaturation studies using circular dichroism. SS16ox strongly interacted with N40, with an equilibrium constant, KD, that was 7-fold higher than wild-type. The association rate constant, kon, of SS16ox was 8.7-fold higher than that of wild-type. This strong interaction can be explained by the entropic effect of the disulfide bond. The introduction of the disulfide bond into C16 stabilizes the beta-hairpin structure of C16, accelerates the association rate with N40, and then stabilizes the whole complex. These results support a hypothetical folding mechanism of protein G where the amphipathic beta-hairpin structure of C16 acts as a nucleus and accelerates folding of the whole molecule.     

Isolation and characterization of a 58-kDa membrane- and microfilament-associated protein from ascites tumor cell microvilli

A 58-kDa protein is found in microvilli and in actin-containing transmembrane complexes of 13762 ascites tumor cells with immobile surface receptors; it is absent from sublines with mobile receptors. 58-kDa protein has been proposed to stabilize microvilli and restrict receptor mobility by stabilizing membrane-microfilament interactions. Antibodies against 58-kDa protein were blot-purified from antisera of rabbits injected with crude transmembrane complex and were used to monitor purification of the protein. 58-kDa protein was extracted from EDTA/EGTA-stripped microvillar microfilament cores with 1 M NaCl. A single depolymerization-polymerization cycle of the microfilaments, followed by solubilization of 58-kDa protein in 1 M NaCl and chromatography on hydroxyapatite-Sephadex G-150, purified the protein to greater than 95% homogeneity. The native molecular weight and frictional coefficient indicated a monomeric, asymmetric structure. 58-kDa protein bound F-actin in pelleting assays and inhibited polymerization of pyrenyl-actin. It also bound phosphatidylserine, phosphatidylinositol, and phosphatidylcholine vesicles in pelleting studies. Immunoblot analyses of endogenously and exogenously proteolyzed microvilli and their membranes and microfilament cores showed specific membrane and microfilament binding fragments of 28-30 kDa. The microfilament- and phospholipid-binding properties of 58-kDa protein and the localization of its proteolysis products are consistent with its proposed role in stabilizing membrane-microfilament interactions in the ascites cell microvilli.     

Isolation, purification and partial characterization of a 30-kDa chlorophyll-a/b-binding protein from spinach

A 30-kDa chlorophyll-a/b-binding protein was purified from photosystem II membrane fragments using Ca(2+)-chelating Sepharose 6B chromatography. The protein binds approximately four chlorophyll a molecules, one chlorophyll b molecule and carotenoids. Its 77-K fluorescence-emission spectrum exhibits a maximum at 680 +/- 1 nm. The protein has a high tendency to form a dimer in the presence of Ca2+.Ca2+ binding affects the low-temperature fluorescence-emission maximum, leading to a decrease in its intensity and a blue shift of 1 nm. Similar spectral changes were obtained in the presence of Mg2+, possibly indicating a common binding domain for both cations. We interpret these observations as cation-induced conformational changes of the protein, which were reversible upon subsequent incubation in EDTA. Evidence is presented for the involvement of carboxyl groups in the coordination sphere of the bivalent cations. The possible structural and functional role of the protein is discussed.     

Isolation and characterization of a full-length cDNA encoding the 55-kDa rabbit zona pellucida protein.

A full-length cDNA (rc55) encoding the major rabbit zona pellucida (ZP) glycoprotein (55 kDa) has been cloned and sequenced. A lambda gt11 expression library was constructed using poly(A)+ mRNA isolated from sexually immature rabbit ovaries which contain large numbers of developing follicles. The rc55 cDNA was identified using affinity purified polyclonal antibodies specific to ZP antigens which are shared among mammalian species. The deduced amino acid sequence of the full-length rc55 clone was matched to the NH2-terminal 25-amino acid sequence obtained for this protein. The predicted amino acid sequence consists of 540 amino acids including a putative signal peptide of 18-24 residues and six potential N-glycosylation sites. The cDNA hybridizes to a 2000-base species of mRNA from rabbit ovary which is not detected in other rabbit tissues. The message is present early in ovarian follicular development and is approximately 600-fold greater in sexually immature as compared with sexually mature rabbit ovaries. This cDNA was expressed as a cro-beta-galactosidase fusion protein using the pEX expression vector. Antibodies against native rabbit ZP, affinity-purified on the recombinant 55-kDa ZP protein, were found to recognize the native rabbit ZP glycoprotein, indicating partial conservation of native epitopes in the expressed recombinant protein.     

Identification and characterization of a mammalian 14-kDa phosphohistidine phosphatase.

Protein histidine phosphorylation in eukaryotes has been sparsely studied compared to protein serine/threonine and tyrosine phosphorylation. In an attempt to rectify this by probing porcine liver cytosol with the phosphohistidine-containing peptide succinyl-Ala-His(P)-Pro-Phe-p-nitroanilide (phosphopeptide I), we observed a phosphatase activity that was insensitive towards okadaic acid and EDTA. This suggested the existence of a phosphohistidine phosphatase different from protein phosphatase 1, 2A and 2C. A 1000-fold purification to apparent homogeneity gave a 14-kDa phosphatase with a specific activity of 3 micro mol.min-1.mg-1 at pH 7.5 with 7 micro m phosphopeptide I as substrate. Partial amino-acid sequence determination of the purified porcine enzyme by MS revealed similarity with a human sequence representing a human chromosome 9 gene of hitherto unknown function. Molecular cloning from a human embryonic kidney cell cDNA-library followed by expression and purification, yielded a protein with a molecular mass of 13 700 Da, and an EDTA-insensitive phosphohistidine phosphatase activity of 9 micro mol.min-1.mg-1 towards phosphopeptide I. No detectable activity was obtained towards a set of phosphoserine-, phosphothreonine-, and phosphotyrosine peptides. Northern blot analysis indicated that the human phosphohistidine phosphatase mRNA was present preferentially in heart and skeletal muscle. These results provide a new tool for studying eukaryotic histidine phosphorylation/dephosphorylation.     

Subfragments from the Fc fragment of human immunoglobulin G. Isolation and physicochemical characterization

A fragment termed fragment Fc' and a related fragment termed fragment pFc' produced by the actions of papain and pepsin respectively on human immunoglobulin G have been isolated and characterized. Amino acid analyses and experiments utilizing cyanogen bromide to cleave the methionyl bonds of the Fc' and pFc' fragments make it possible to locate both fragments within the known chain structure of the immunoglobulin G molecule. The pFc' fragment is probably a non-covalently linked dimer situated at the C-terminal end of the molecule, containing about 232 amino acid residues and having a molecular weight of 26000. The Fc' fragment is a similar dimer of about 182 residues extending from near residue 14 to near residue 105 (numbered from the C-terminal end) of the gamma-chain and has a molecular weight of 21000.     

Isolation and immunological characterization of the mammalian 32-/34-kDa stress protein

Challenge of mammalian cells with heavy metals or sulfhydryl-reactive agents including sodium arsenite induces the de novo synthesis of a 32-/34-kDa stress protein (p32) (M. M. Caltabiano, T. P. Koestler, G. Poste, and R. G. Greig (1986) J. Biol. Chem. 261, 13,381). Here we report that antibody prepared against p32/p34 purified from human A375 melanoma cells immunoprecipitated an antigen of similar molecular mass from a panel of human, rat, and murine cells following challenge with sodium arsenite. No reactivity was observed in lysates from control, uninsulted cultures. The precise molecular mass of the arsenite-induced antigen was species-specific: 32 kDa (human and rat) and 34 kDa (murine). Indirect immunofluorescence analysis using affinity-purified monospecific IgG demonstrated that p32/p34 was localized to the cytoplasm and displayed a perinuclear distribution.     

Isolation and characterization of a fragment of rat thyroglobulin gene

A rat genomic library was screened for thyroglobulin gene clones with recombinant plasmids containing rat thyroglobulin complementary DNA inserts. Two identical recombinant phages were found. A map of the inserted genomic sequence established by restriction and blotting experiments, and electronic microscopy revealed that this fraction of the gene was extensively split. Exons were ≤ 200 base pair long while the introns represented 93% of the insert. A fragment subcloned in plasmid pBR 322 was shown to contain repetitive sequences when used in Southern blot experiments with rat total genomic DNA.     

Isolation and partial characterization of a 48-kDa protein which is induced in normal lymphocytes upon mitogenic stimulation

A 48-kDa protein (p48) crossreactive with an antiserum directed against the 12 C-terminal amino acids of the human cellular myc gene-encoded protein was isolated from a Burkitt lymphoma cell line. The p48 protein is a basic protein and has a cytoplasmic localization. An antiserum prepared against purified p48 reacts specifically with a 48-kDa protein present in a variety of mouse and human cells. This polypeptide is detected at very low levels in normal, resting, peripheral blood lymphocytes, but is induced several-fold by stimulation with either concanavalin A or pokeweed mitogen. The association of p48 induction with a proliferative response and the crossreactivity with the cellular myc protein are discussed.     

Isolation and characterization of the cell-associated region of group A streptococcal M6 protein.

DNA sequence analysis of the complete M6 protein gene revealed 19 hydrophobic amino acids at the C terminus which could act as a membrane anchor and an adjacent proline- and glycine-rich region likely to be located in the cell wall. To define this region within the cell wall and its role in attaching the molecule to the cell, we isolated the cell-associated fragment of the M protein. Assuming that the cell-associated region of the M protein would be embedded within the wall and thus protected from trypsin digestion, cells were digested with this enzyme, and the wall-associated M protein fragment was released by phage lysin digestion of the peptidoglycan. With antibody probes prepared to synthetic peptides of C-terminal sequences, a cell wall-associated M protein fragment (molecular weight, 16,000) was identified and purified. Amino acid sequence analysis placed the N terminus of the 16,000-molecular-weight fragment at residue 298 within the M sequence. Amino acid composition of this peptide was consistent with a C-terminal sequence lacking the membrane anchor. Antibody studies of nitrous acid-extracted whole bacteria suggested that, in addition to the peptidoglycan-associated region, a 65-residue helical segment of the C-terminal domain of the M protein is embedded within the carbohydrate moiety of the cell wall. Since no detectable amino sugars were associated with the wall-associated fragment, the C-terminal region of the M6 molecule is likely to be intercalated within the cross-linked peptidoglycan and not covalently linked to it. Because the C-terminal region of the M molecule is highly homologous to the C-terminal end of protein A from staphylococci and protein G from streptococci, it is likely that the mechanism of attachment of these proteins to the cell wall is conserved.     

链球菌蛋白G的构建、重组表达及鉴定

化学合成链球茵蛋白G的C3D基因片段,通过分子生物学的方法对蛋白G（proteinG）的C3片段进行PCR扩增,拼接形成含有两个和三个重复C3片段的重组链球茵蛋白G,C3片段间以链接区D连接,即形成C3DC3和C3DC3DC3的形式,进而克隆到质粒pET21中,在大肠杆菌BL21（DE3）中表达。重组表达的蛋白经过DEAE—Sepharose和IgG—Sepharose纯化,得到纯化的重组蛋白。采用非竞争性酶免疫法对重组蛋白与不同来源IgG的结合常数进行测定,实验结果显示两种重组链球茵蛋白G均可有效地与小鼠、兔及山羊等多种不同来源抗体特异性结合。这些实验结果为下一步研究奠定了基础。     

Construction, expression and identification of a recombinant streptococcal protein G

化学合成链球菌蛋白G的C3D基因片段,通过分子生物学的方法对蛋白G(proteinG)的C3[1]片段进行PCR扩增,拼接形成含有两个和三个重复C3片段的重组链球菌蛋白G,C3片段间以链接区D连接,即形成C3DC3和C3DC3DC3的形式,进而克隆到质粒pET21中,在大肠杆菌BL21(DE3)中表达.重组表达的蛋白经过DEAE - Sepharose和IgG- Sepharose纯化,得到纯化的重组蛋白.采用非竞争性酶免疫法对重组蛋白与不同来源IgG的结合常数进行测定,实验结果显示两种重组链球菌蛋白G均可有效地与小鼠、兔及山羊等多种不同来源抗体特异性结合.这些实验结果为下一步研究奠定了基础.     

Expression and purification of a truncated recombinant streptococcal protein G.

The gene for Protein G from Streptococcus strain G148 was cloned and expressed in Escherichia coli. The regions on the gene corresponding to the albumin-binding domains and the Fab-binding region were then deleted by site-directed mutagenesis. The translation of regions corresponding to the cell-wall- and membrane-binding domains was prevented by introduction of stop codons upstream of these domains. This recombinant DNA sequence codes for a protein (G') that contains repetitive regions and that binds only the Fc portion of IgG, analogously to Protein A. Translation of the sequence produces a protein with an Mr of about 20,000. The nucleotide sequence differs from those published previously [Guss, Eliasson, Olsson, Uhlén, Frej, Jornvall, Flock & Lindberg (1986) EMBO J. 5, 1567-1575; Olsson, Eliasson, Guss, Nilsson, Hellman, Lindberg & Uhlén (1987) Eur. J. Biochem. 168, 319-324]. The protein can be substantially purified on a large scale by chromatography on IgG-Sepharose 4B. Homogeneous Protein G' can be prepared by anion-exchange f.p.l.c. on Mono Q HR. This Protein G' has a pI of 4.19 and SDS/PAGE gives an apparent anomalous Mr of 35,000.     

Crystallization and preliminary X-ray analysis of the complex between a mouse Fab fragment and a single IgG-binding domain from streptococcal protein G.

The Fab fragment of a mouse immunoglobulin G1, complexed with a single IgG-binding domain from streptococcal protein G, has been crystallized in a form suitable for analysis by X-ray diffraction. The needle-shaped crystals were grown from polyethylene glycol 4000 using vapour diffusion methods and diffract to 2.3 A resolution. The space group is P2(1)2(1)2(1) (a = 64.5 A, b = 70.5 A and c = 120.1 A), with one Fab-protein G domain complex in the asymmetric unit. Solution of the three-dimensional structure of the complex will permit a detailed analysis of the molecular interactions between protein G and the Fab portion of IgG.