Streptococcal protein G, expressed by streptococci or by Escherichia coli, has separate binding sites for human albumin and IgG

Protein G is expressed at the cell surface of certain group C and group G streptococcal strains. The protein shows a unique and specific affinity for the Fc region of mammalian polyclonal and monoclonal immunoglobulin G (IgG). We have cloned the streptococcal gene coding for protein G into E. coli, using phage lambda as the vector. The protein G produced by E. coli infected with this phage was detected and analysed in Western blot experiments using radiolabelled IgG Fc fragments as a probe. Three major IgG Fc-binding bands were obtained corresponding to apparent mol. wts of 47,000, 57,000 and 65,000, respectively. Analysis of the expression in E. coli indicates that this heterogeneity is caused by a post-translational degradation of the molecule before lysis of the lambda infected E. coli cells occurred. The protein G produced in E. coli was purified by affinity chromatography on IgG-Sepharose followed by gel-filtration on Sephadex G-200. This highly purified E. coli-produced protein G was compared to protein G solubilized by papain from streptococci, in direct binding experiments and in a competitive binding assay. The two protein G variants were found to interact with polyclonal IgG from different species in a similar way. Streptococcal strains expressing protein G also show affinity for human albumin, and at the molecular level protein G was found to be responsible also for the binding of albumin. Thus, both E. coli-produced protein G and the proteolytic fragment of protein G obtained from streptococci, bound albumin. On the protein G molecule, two different and separate sites were found to bind IgG and albumin. Finally, when whole streptococci were incubated with human plasma, the interactions with protein G caused a coating of the bacteria with albumin and IgG, whereas other plasma proteins showed no affinity for protein G.     

Cleaning procedure for protein G affinity columns

An efficient method for cleaning protein G affinity columns is described in which impurities are removed effectively with a combination of 4 mol/l urea and 0.1 mol/l sodium hydroxide. These cleaning conditions have only a minor deleterious effect on the binding capacity of the column and no effect on its selectivity for IgG. This cleaning procedure should prolong the life of costly affinity columns used in the purification of immunoglobulin G (IgG).     

Selective affinity of protein A containing staphylococci for monomeric and polymeric IgG

Protein A containing staphylococci were saturated with human monomeric IgG (mIgG) and cross-linked with glutaraldehyde. The resulting material (SMG) preferentially bound aggregated IgG (aIgG) and soluble immune complexes (CIC). One milliliter of a 10% suspension of SMG bound approximately 30 micrograms of mIgG and 1000 micrograms of aIgG and CIC. The binding of aIgG to SMG was reduced to approximately 50% at a 20-fold excess of mIgG over aIgG. CIC and aIgG could be released from SMG by elution with 3 M KSCN. The results indicate that SMG can be used for identification and removal of CIC in patient plasma.     

A novel method to clean protein G agarose for affinity column matrix with renewed binding capacity and high IgG selectivity

Attempts have been made at finding ways of cleaning used protein G agarose to revive their efficiency and make them reusable for purifying immunoglobulin G (IgG) in affinity column chromatography. A successful cleaning procedure that involved the use of 4-mol/L urea with 0.1-mol/L sodium hydroxide has been previously evaluated although it had some deleterious effect on the column binding capacity and compromised the resin efficiency. Efforts to develop base-tolerant affinity columns by using genetically engineered ligands such as the recombinant protein A from GE Healthcare have achieved some progress, with the column efficiency getting compromised to a lesser extent. However, genetically engineered ligands are even more expensive and may not be readily affordable in modest laboratory settings, especially if large-scale purchases are needed in routine use. We report here a novel and simple cleaning method involving the use of polyethylene glycol (PEG8000) that renews matrix-binding capacity comparable to a new resin while retaining high selectivity for IgG.     

Isolation of subclasses of human IgG with affinity chromatography

A method is described for the isolation of subclasses of human IgG. Two myeloma proteins, after isolation by DEAE-Sephadex chromatography, were further purified on subclass-specific anti-IgG1 and 2 Sepharose 4B columns. Contaminating IgG1 and 2 were removed from the isolated myeloma proteins.     

Immobilization staphylococcal protein a on magnetic cellulose microspheres for IgG affinity purification

The main objective of this study is to develop and evaluate the immobilization of Staphylococcal Protein A on magnetic cellulose microspheres (SPA-MCMS) for immunological capture of IgG. After cloning, expression and separation, SPA was immobilized onto MCMS to prepare a magnetic affinity media subject to the purification of IgGs. The binding capacity, binding time, leakage of SPA and its reproducibility were optimized to improve the binding efficiency with an appropriate amount and recovery of IgG. Rabbit IgG was successfully purified from serum in a single-step by SPA-MCMS with an overall recovery of 73.18% and purity of 90.27%. Therefore, this study effectively illustrated the advantages of magnetic microcarriers for rapid and efficient purification of antibodies. The separation media shows a high potential for the future development of affinity isolation and immunodiagnostic application.     

Streptococcal protein G: A sensitive tool for detection of antibodies to human immunodeficiency virus proteins in Western blot analysis

Protein G is a cell wall protein of group C and G streptococci which binds human IgG antibodies of all four subclasses with high affinity. This property of the molecule was utilized to develop a sensitive Western blot assay to detect antibodies against HIV proteins in patient sera.     

Rat IgG subclasses: Differences in affinity to protein A-Sepharose

The binding of rat IgG subclasses: IgG1, IgG2a, IgG2b and IgG2c to staphylococcal protein A has been investigated by fractionation of monoclonal rat IgG or normal rat serum on protein A-Sepharose. Elution was performed by buffers of decreasing pH (from 9.0 to 3.0) or by sodium thiocyanate gradients (NaSCN) at pH 7.4. Differences in affinity were found in part to be related to the subclass with the order: IgG2c > IgG1 > IgG2b > IgG2a (the affinity of IgG2b being very weak, and IgG2a having no affinity for protein A). Differences in affinity between monoclonal IgG2cs were also demonstrated, some being eluted at pH 4.0-3.0 (or 1 M NaSCN) and others at pH 6.0 (or 0.5 M NaSCN). Each monoclonal IgGl also exhibited heterogeneity with fractions eluted at pH 8.0,7.0 and 6.0. The elution pattern of IgG subclasses from normal rat serum was found to be in agreement with the results obtained from monoclonal IgG. Pure IgG1 and IgG2c were recovered from rat serum at pH 7.0 and 4.0–3.0 respectively.     

Differential binding characteristics of protein G and protein A for Fc fragments of papain-digested mouse IgG

It has been previously reported that staphylococcal protein A (SPA) bound only to the Fc region of mouse immunoglobulin G (IgG) and streptococcal protein G (SPG) bound to both Fab and Fc regions of mouse IgG and the binding sites for SPG and SPA on Fc were overlapped. In this study the binding characteristics of SPG and SPA for papain-digested mouse IgG were analysed. Papain digestion of mouse IgG purified from CAy-IFNg99C hybridoma (secreting IgG1 monoclonal antibody specific for human interferon gamma)-induced ascites resulted in Fab and two major Fc fragments referred to as the high molecular weight (HMW) and the low molecular weight (LMW) Fc fragments. SPG bound to Fab and the LMW Fc fragments of the papain-digested IgG. However SPG did not bind to the HMW Fc fragment. SPA showed practically no reactivity with the Fab and the LMW Fc fragments of the papain-digested mouse IgG but only to the HMW Fc fragment. SPG and SPA binding assays showed that papain digestion discriminated the SPG and SPA binding sites in the Fc fragment of mouse IgG. These results demonstrated a clear evidence for the presence of two independent SPG and SPA binding sites in the Fc fragment of mouse IgG.     

Purification of immunoglobulins G by protein A/G affinity membrane chromatography

An affinity membrane grafted with protein A/G or protein A was characterized for human and mouse immunoglobulins G purification. Breakthrough curves up to ligand saturation were measured and used to study the effects of flow velocities, feed solution concentrations and protein A/G versus protein A membranes. Increased flow-rate did not decrease the amount of IgG bound to the membranes. Increased feed solution concentration allowed more IgG to bind prior to breakthrough. Kinetic parameters for immunoglobulins G sorption to immobilized protein A were measured in batch experiments. The static binding capacity was determined to be 6.6 mg ml(-1) membrane volume. Finally, this affinity membrane was used to purify IgG from cell culture supernatant. The electrophoresis of the purified IgG fractions did not show any contaminant.     

Activation of human mast cells through the high affinity IgG receptor

Mast cells are known to participate in the induction of inflammation through interaction of antigen with specific IgE bound to the high affinity receptor for IgE (FcepsilonRI). Human mast cells, derived from CD34(+) hematopoietic precursors, not only express FcepsilonRI but also express high affinity receptors for IgG (FcgammaRI), the latter only after IFN-gamma exposure. Human mast cells that express FcgammaRI are activated following FcgammaRI aggregation, either using antibodies directed to the receptor or through IgG bound to the receptor. This activation results in degranulation, with the release of granule-associated mediators, and the generation of metabolites of arachidonic acid and secretion of chemokines and cytokines. These observations provide evidence that human mast cells may also be recruited into inflammation through IgG-dependent mechanisms.     

Chromatographic behavior of mouse serum immunoglobulin G in protein G perfusion affinity chromatography

In this study chromatographic behavior of mouse serum immunoglobulin G (IgG) on a protein G perfusion affinity chromatographic column was investigated experimentally. The results indicate that the protein G column has no non-specific binding to the other proteins in mouse serum but an irreversible adsorption to IgG under the conditions investigated. It was found that variations of the elution solution composition, ionic strength and pH played to some extent an essential effect on the chromatographic behavior of IgG. The influence of the mobile phase flow-rate on the chromatographic behavior of IgG was also researched. These results show that the dissociation of IgG from protein G affinity packings becomes the rate-limiting step in the perfusion affinity chromatographic separation process.     

Isolation of immunoglobulin G by affinity chromatography using an IgG Fc receptor protein from Streptococcus dysgalactiae coupled to a solid phase

A culture of Streptococcus dysgalactiae (C 26) was shown to bind only to 125I-IgG, whereas another S. dysgalactiae culture (C 12) bound both 125I-IgG and 125I-albumin. The IgG-binding proteins could be readily solubilized by lysozyme treatment of the bacteria and isolated by affinity chromatography on IgG Sepharose. The purified IgG-binding protein from S. dysgalactiae C 26, which lacked simultaneous albumin binding activity, precipitated with IgG preparations from man, cow, horse, pig and mouse but not with chicken IgG. This IgG-binding protein was coupled to CNBr-activated Sepharose and subsequently used for the purification of IgG from both bovine and human serum. SDS-PAGE and immunoelectrophoretic studies confirmed the purity of the eluted proteins.     

Preparation of human sera containing one single IgG subclass using affinity chromatography

Monoclonal antibodies reactive against one or three human IgG subclasses were immobilised on agarose beads, put into columns and used in combination with coupled polyclonal anti-IgA and anti-IgM reactive gels to obtain serum preparations devoid of IgA, IgM and all but one IgG subclass. This was possible after one single run over the appropriate combination of columns; only for IgG4 preparations was a second purification step sometimes required to reach purity. Negative affinity chromatography was used throughout, thus the resulting preparations had not been exposed to high ionic strength conditions, non-physiological pH buffers or chaotropic agents in concentrations ordinarily used to elute bound material from affinity columns. The yield was approximately 50% and 20-30% if two runs were required. Regeneration of the columns permitted repetitive use, so far up to 30 times without substantial loss of activity. The protocol offers an easy, comparatively fast and reproducible method to obtain human serum preparations containing only IgG1,2,3, or 4.     

Escherichia coli do not express Fc-receptors for human immunoglobulin G (IgG)

Gram-positive bacterial pathogens express immunoglobulin (Ig) binding proteins that perturb Fc-dependent functions such as the interaction with complement or phagocytic Fc-receptors. The possession of such molecules by gram-negative bacteria, including Escherichia coli (E. coli), has also been documented. In many such studies, the detection of Ig binding has relied on the use of polyclonal antibodies as detecting reagents. These are not ideal since such preparations may be contaminated with E. coli specific IgG, allowing for the potential misinterpretation of specific F(ab')(2) binding as non-specific Fc mediated binding. Here we use mono-specific recombinant antibodies to develop a novel assay for Ig binding non reliant on traditional polyclonal antibodies, allowing us to demonstrate the unequivocal absence of Fc binding proteins from E. coli.     

Properties of Human Anti-Group B Streptococcal Type III Capsular IgG Antibody

The group B streptococcus is the commonest cause of bacterial infection in the newborn. In an attempt to prevent these infections, various vaccines are in development, most of which contain at least one of the capsular carbohydrates of the bacterium. We present new detailed data on the natural human antibody response to the type III capsular carbohydrate as we believe it is important to ascertain equivalent data for any new candidate vaccine in order to predict efficacy. We demonstrate that naturally occurring IgG is opsonically active in a complement-dependent manner, that fractions of differing avidity isolated from single donors have broadly similar opsonic activity, that the clonotypes from four individuals are restricted in number to a maximum of 15, and that binding kinetics ascertained using a resonant mirror biosensor show that specific antibodies have a moderately high affinity (meanK_d= 1.1e-8 M).     

Development of a rapid, single-step procedure using protein G affinity chromatography to deplete fetal calf serum of its IgG and to isolate murine IgG1 monoclonal antibodies from supernatants of hybridoma cells

Fetal calf serum (FCS) was depleted of its immunoglobulin G (IgG) in a rapid procedure using protein G affinity chromatography. 20 ml of FCS was depleted of its IgG in less than 80 min by applying 5 ml of FCS to a 1 ml HiTrap protein G Sepharose column followed by appropriate elution. Various concentrations of IgG-depleted FCS (G-FCS) were used in RPMI-1640 medium to grow the mouse hybridoma cell lines CAy-G (anti-HBs IgG1 mAb producing hybridoma cell) and CAy-M (anti-HBs IgM mAb producing hybridoma cell), which secreted hepatitis B virus surface antigen (HBsAg)-reactive IgG1 and IgM monoclonal antibodies (mAbs), respectively. Antibody production and cell growth were used as indices to compare the efficacy of RPMI/G-FCS with that of RPMI/FCS and serum/protein-free Hybri Max (Sigma, MO, USA) hybridoma medium. MAb production and cell growth of CAy-G and CAy-M hybridoma cell lines in RPMI/G-FCS were similar to culture in RPMI/FCS and significantly better than culture in Hybri Max. We found that G-FCS was superior to whole FCS as a culture supplement for the purification of IgG1 mAbs. IgG1 mAbs were isolated in a single-step procedure using protein G affinity chromatography, from the supernatant of CAy-G hybridoma cells cultured in RPMI/10% G-FCS (RPMI-1640 medium supplemented with 10% G-FCS). SDS-PAGE analysis revealed that the purity of IgG isolated from the supernatant of CAy-G cells cultured in RPMI/10% G-FCS was more than 99%.     

Immunoglobulin G has a role for systemic protein modulation in vivo: a new concept of protein homeostasis

The constant level of various proteins including albumin and cellular components in intravascular pool in vivo is strictly controlled by an unknown homeostatic mechanism, although there are fluctuations seen in pathologic conditions. Because the majority of the IgG in the serum is regarded as self-reactive natural autoantibodies, IgG may have a role to react with all proteins in vivo. It is hypothesized that like an immune system, a homeostatic mechanism for the protein pool also has a sensitive role to identify and memorize the extent and repertoire of both normal and pathogenic proteins on an individual basis, and IgG may be one of the major players in performing these functions. This hypothesis may explain the unresolved clinical observations as followed: (1) the marked increased IgG levels observed in self-limiting diseases presumed to come from immunological insults such as acute poststreptococcal glomerulonephritis and Kikuchi-Fujimoto disease, (2) an immediate reduction of all protein levels except immunoglobulins after intravenous immunoglobulin (IVIG) treatment in Kawasaki disease, (3) a unified explanation for the variety of immunomodulating effects exerted by IVIG, (4) the IgG-enzyme complexes observed in benign conditions such as macroamylasemia and hyperphosphatasemia, and (5) the marked decreased IgG level, which is correlated with the albumin level in minimal change nephrotic syndrome. IgG may be a 'watch-dog' for the disturbances of protein homeostasis in vivo. IgG may control the pathogenic proteins that appeared in disordered states, and it may help prevent the loss of proteins in case of nephrotic syndrome.     

Lipids are co-eluted with immunoglobulins G during purification by recombinant streptococcal protein G affinity chromatography

The efficiency of recombinant streptococcal protein G (rec-spG) affinity column chromatography in purifying immunoglobulins G (IgG) from lipids has been studied, with particular reference to IgG fractions from bronchoalveolar lavage (BAL) fluid and serum samples from different sources. It was found that the IgG fractions purified by rec-spG affinity column chromatography also contained cholesterol and phospholipids.