Improving the tolerance of a protein a analogue to repeated alkaline exposures using a bypass mutagenesis approach

Staphylococcal protein A (SPA) is a cell surface protein expressed by Staphylococcus aureus. It consists of five repetitive domains. The five SPA-domains show individual interaction to the Fc-fragment as well as certain Fab-fragments of immunoglobulin G (IgG) from most mammalian species. Due to the high affinity and selectivity of SPA, it has a widespread use as an affinity ligand for capture and purification of antibodies. One of the problems with proteinaceous affinity ligands in large-scale purification is their sensitivity to alkaline conditions. SPA however, is considered relatively stable to alkaline treatment. Nevertheless, it is desirable to further improve the stability in order to enable an SPA-based affinity medium to withstand even longer exposure to the harsh conditions associated with cleaning-in-place (CIP) procedures. For this purpose, a protein engineering strategy, which was used earlier for stabilization and consists of replacing the asparagine residues, is employed. Since Z in its "nonengineered" form already has a significant tolerance to alkaline treatment, small changes in stability due to the mutations are difficult to assess. Hence, in order to enable detection of improvements regarding the alkaline resistance of the Z domain, we chose to use a bypass mutagenesis strategy using a mutated variant Z(F30A) as a surrogate framework. Z(F30A) has earlier been shown to possess an affinity to IgG that is similar to the wild-type but also demonstrates decreased structural stability. Since the contribution of the different asparagine residues to the deactivation rate of a ligand is dependent on the environment and also the structural flexibility of the particular region, it is important to consider all sensitive amino acids one by one. The parental Z-domain contains eight asparagine residues, each with a different impact on the alkaline stability of the domain. By exchanging asparagine 23 for a threonine, we were able to increase the stability of the Z(F30A) domain in alkaline conditions. Also, when grafting the N23T mutation to the Z scaffold, we were able to detect an increased tolerance to alkaline treatment compared to the native Z molecule.     

Engineering streptococcal protein G for increased alkaline stability

Most protein-based affinity chromatography media are very sensitive towards alkaline treatment, which is a preferred method for regeneration and removal of contaminants from the purification devices in industrial applications. In a previous study, we concluded that a simple and straightforward strategy consisting of replacing asparagine residues could improve the stability towards alkaline conditions. In this study, we have shown the potential of this rationale by stabilizing an IgG-binding domain of streptococcal protein G, i.e. the C2 domain. In order to analyze the contribution of the different amino acids to the alkaline sensitivity of the domain we used a single point mutation strategy. Amino acids known to be susceptible towards high pH, asparagine and glutamine, were substituted for less-alkali-susceptible residues. In addition, aspartic acid residues were mutated to evaluate if the stability could be further increased. The stability of the different C2 variants was subsequently analyzed by exposing them to NaOH. The obtained results reveal that the most sensitive amino acid towards alkaline conditions in the structure of C2 is Asn36. The double mutant, C2(N7,36A), was found to be the most stable mutant constructed. In addition to the increased alkaline stability and also very important for potential use as an affinity ligand, this mutated variant also retains the secondary structure, as well as the affinity to the Fc fragment of IgG.     

Determination of the solution structures of domains II and III of protein G from Streptococcus by 1H nuclear magnetic resonance.

We have used 1H nuclear magnetic resonance spectroscopy to determine the solution structures of two small (61 and 64 residue) immunoglobulin G (IgG)-binding domains from protein G, a cell-surface protein from Streptococcus strain G148. The two domains differ in sequence by four amino acid substitutions, and differ in their affinity for some subclasses of IgG. The structure of domain II was determined using a total of 478 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints; that of domain III was determined using a total of 445 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints. A protocol which involved distance geometry, simulated annealing and restrained molecular dynamics was used to determine ensembles of 40 structures consistent with these restraints. The structures are found to consist of an alpha-helix packed against a four-stranded antiparallel-parallel-antiparallel beta-sheet. The structures of the two domains are compared to each other and to the reported structure of a similar domain from a protein G from a different strain of Streptococcus. We conclude that the difference in affinity of domains II and III for IgG is due to local changes in amino acid side-chains, rather than a more extensive change in conformation, suggesting that one or more of the residues which differ between them are directly involved in interaction with IgG.     

pH-sensitive interactions between IgG and a mutated IgG-binding protein based upon two B domains of protein A from Staphylococcus aureus.

A fusion protein, consisting of the N-terminal 81 amino acids from an inactive bovine DNase I (Q38,E39-E38,Q39) and two sequential synthetic IgG-binding domains based upon domain B of Protein A from Staphylococcus aureus has been shown to bind to porcine IgG with a similar affinity and pH profile to Protein A. The same residue in each B domain (Tyr111 and Tyr169) has been mutated by cassette mutagenesis to Ser, Glu, His, Lys or Arg and the effect of the mutation on binding interactions with porcine IgG investigated. The evidence presented suggests that the interactions at the B domain are highly sensitive to the presence of a charged residue.     

A biomimetic Protein G affinity adsorbent: an Ugi ligand for immunoglobulins and Fab fragments based on the third IgG‐binding domain of Protein G

This work reports the development of a synthetic affinity adsorbent for immunoglobulins based on the Fab‐binding domain of Streptococcal Protein G (SpG‐domain III). The ligand (A2C7I1) was synthesized by the four‐component Ugi reaction to generate a substituted peptoidal scaffold mimicking key amino acid residues of SpG. Computer‐aided analysis suggests a putative binding site on the C_H1 domain of the Fab molecule. In silico studies, supported by affinity chromatography in comparison with immobilized SpG, as well as analytical characterization by liquid chromatography/electrospray ionization–mass spectrometry and ¹H nuclear magnetic resonance of the ligand synthesized in solution, indicated the authenticity and suitability of the designed ligand for the purification of immunoglobulins. The immobilized ligand displayed an apparent static binding capacity of ~17 mg IgG ml^?1 and a dissociation constant of 5.34 × 10^?5 M. Preparative chromatography demonstrated the ability of the immobilized ligand to purify IgG and Fab fragments from crude mammalian and yeast cell cultures, under near physiological ionic strength and pH, to yield proteins of 99% and 93% purity, respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy

The Fc portion of immunoglobulin G (IgG) expresses the biantennary complex type oligosaccharides at Asn297 of the C(H)2 domain of each heavy chain with microheterogeneities depending on physiological and pathological states. These N-glycans are known to be essential for promotion of proper effector functions of IgG such as complement activation and Fcgamma receptor (FcgammaR)-mediated activities. To gain a better understanding of the role of Fc glycosylation, we prepared a series of truncated glycoforms of human IgG1-Fc and analyzed their interactions with human soluble FcgammaRIIIa (sFcgammaRIIIa) and with staphylococcal protein A by surface plasmon resonance and nuclear magnetic resonance (NMR) methods. Progressive but less pronounced reductions in the affinity for sFcgammaRIIIa were observed as a result of the galactosidase and subsequent N-acetylhexosaminidase treatments of IgG1-Fc. The following endoglycosidase D treatment, giving rise to a disaccharide structure composed of a fucosylated GlcNAc, abrogated the affinity of IgG1-Fc for sFcgammaRIIIa. On the other hand, those glycosidase treatments did not significantly affect the affinity of IgG1-Fc for protein A. Inspection of stable-isotope-assisted NMR data of a series of Fc glycoforms indicates that the stepwise trimming out of the carbohydrate residues results in concomitant increase in the number of amino acid residues perturbed thereby in the C(H)2 domains. Furthermore, the cleavage at the GlcNAcbeta1-4GlcNAc glycosidic linkage induced the conformational alterations of part of the lower hinge region, which makes no direct contact with the carbohydrate moieties and forms the major FcgammaR-binding site, while the conformation of the C(H)2/C(H)3 interface was barely perturbed that is the protein A-binding site. These results indicate that the carbohydrate moieties are required for maintaining the structural integrity of the FcgammaR-binding site.     

Crystallographic and mutational studies of Mycobacterium tuberculosis recA mini-inteins suggest a pivotal role for a highly conserved aspartate residue

The 440 amino acid Mtu recA intein consists of independent protein-splicing and endonuclease domains. Previously, removal of the central endonuclease domain of the intein, and selection for function, generated a 168 residue mini-intein, DeltaI-SM, that had splicing activity similar to that of the full-length, wild-type protein. A D422G mutation (DeltaI-CM) increased C-terminal cleavage activity. Using the DeltaI-SM mini-intein structure (presented here) as a guide, we previously generated a highly active 139 residue mini-intein, DeltaDeltaI(hh)-SM, by replacing 36 amino acid residues in the residual endonuclease loop with a seven-residue beta-turn from the autoprocessing domain of Hedgehog protein. The three-dimensional structures of DeltaI-SM, DeltaDeltaI(hh)-SM, and two variants, DeltaDeltaI(hh)-CM and DeltaDeltaI(hh), have been determined to evaluate the effects of the minimization on intein integrity and to investigate the structural and functional consequences of the D422G mutation. These structural studies show that Asp422 is capable of interacting with both the N and C termini. These interactions are lacking in the CM variant, but are replaced by contacts with water molecules. Accordingly, additional mutagenesis of residue 422, combined with mutations that isolate N-terminal and C-terminal cleavage, showed that the side-chain of Asp422 plays a role in both N and C-terminal cleavage, thereby suggesting that this highly conserved residue regulates the balance between the two reactions.     

PDZ domains of RseP are not essential for sequential cleavage of RseA or stress‐induced σE activation in vivo

The Escherichia coli σ^E extracytoplasmic stress response monitors and responds to folding stress in the cell envelope. A protease cascade directed at RseA, a membrane‐spanning anti‐σ that inhibits σ^E activity, controls this critical signal‐transduction system. Stress cues activate DegS to cleave RseA; a second cleavage by RseP releases RseA from the membrane, enabling its rapid degradation. Stress control of proteolysis requires that RseP cleavage is dependent on DegS cleavage. Recent in vitro and structural studies found that RseP cleavage requires binding of RseP PDZ‐C to the newly exposed C‐terminal residue (Val148) of RseA, generated by DegS cleavage, explaining dependence. We tested this mechanism in vivo. Neither mutation in the putative PDZ ligand‐binding regions nor even deletion of entire RseP PDZ domains had significant effects on RseA cleavage in vivo, and the C‐terminal residue of DegS‐processed RseA also little affected RseA cleavage. Indeed, strains with a chromosomal rseP gene deleted for either PDZ domain and strains with a chromosomal rseA V148 mutation grew normally and exhibited almost normal σ^E activation in response to stress signals. We conclude that recognition of the cleaved amino acid by the RseP PDZ domain is not essential for sequential cleavage of RseA and σ^E stress response in vivo.     

Biomimetic design of affinity peptide ligands for human IgG based on protein A-IgG complex

Staphylococcal protein A (SpA) has been widely used as an affinity ligand for the purification of immunoglobulin G (IgG). Based on the affinity motif of SpA, we have herein developed a biomimetic design strategy for affinity peptide ligands of IgG. First, according to the distribution of the six hot spots of the SpA affinity motif determined previously, the number of residues that should be inserted into between the hot spots was determined. Cysteine was introduced as one of the middle inserted residues of the peptide for later immobilization. Then, amino acid location was performed to identify other amino acid residues for insertion, leading to the construction of a peptide library. The library was screened by using different molecular simulation protocols, resulting in the selection of 15 peptide candidates. Thereafter, molecular dynamics simulations were performed to validate the dynamics of the affinity interactions between the candidates and IgG, and 14 of them were found to keep high affinities. Finally, the affinity and specificity of the top one ligand FYWHCLDE were exemplified by protein chromatography and IgG purification. The results indicate that the design strategy was successful and the affinity peptide ligand for IgG is promising for application in antibody purifications.     

Identification,characterization, and cloning of an immunoglobulin degrading enzyme in the cat flea,Ctenocephalides felis

The degradation of cat immunoglobulin G (IgG) in blood-fed adult C. felis midguts was examined. SDS-PAGE analysis of dissected midgut extracts obtained from C. felis that had been blood fed for various times between 0 to 44 h revealed that by 24 h most of the high molecular weight proteins, including the heavy chain of IgG, were digested. A 31-kDa serine protease with IgG degrading activity was purified from fed C. felis midguts by benzamidine affinity chromatography, hydrophobic interaction chromatography, and cation exchange chromatography. Three primary cleavage products between 30- and 40-kDa were observed when the purified protease was incubated with protein A purified cat IgG. N-terminal amino acid sequence analysis of the products revealed that the IgG degrading protease cleaves after specific cysteine and lysine residues within the hinge region of IgG. The enzyme is also capable of degrading other immunoglobulins, serum albumin, and hemoglobin, suggesting that it may have roles in both combating the host's immune system and providing nutrients for the flea. A cDNA clone encoding the 265 amino acid IgG degrading protease proenzyme was isolated. When expressed in a baculovirus/insect cell expression system, the recombinant protein had the same N-terminus as the processed 237 amino acid mature native protein and possessed IgG degrading activity indistinguishable from the native protein. Arch. Insect Biochem.     

Isolation and characterization of a 14-kDa albumin-binding fragment of streptococcal protein G

Protein G, a streptococcal cell wall protein, has separate binding sites for human albumin and IgG. Streptococci expressing protein G were treated with the bacteriolytic agent mutanolysin. Several IgG- and human serum albumin (HSA)-binding peptides were identified in the material thus solubilized and one of these, a 14-kDa peptide, was found to bind HSA but not IgG in Western blot experiments. This molecule was purified by affinity chromatography on Sepharose coupled with HSA followed by gel filtration on Sepharose 6B and a final affinity chromatography on IgG-Sepharose, by which low Mr W(15 to 20 kDa)IgG-binding peptides were removed. In different binding experiments the purified 14-kDa peptide bound exclusively HSA and the equilibrium constant between the peptide and HSA was determined to be 3.4 X 10(8) M-1. The relation between the 14-kDa molecule and protein G was studied by analyzing the N-terminal amino acid sequence of the peptide and comparing it with the previously determined protein G sequence. The 40 N-terminal amino acids were found to be identical with an amino acid sequence starting at position 62 in the protein G molecule. These and previous data enabled us to locate the albumin binding to the repetitively arranged domains in the N-terminal half of the protein G molecule.     

Streptococcal protein G. Gene structure and protein binding properties

Protein G was solubilized from 31 human group C and G streptococcal strains with the muralytic enzyme mutanolysin. As judged by the mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the binding patterns of the solubilized protein G molecules in Western blot experiments, the strains could be divided into three groups, represented by the group G streptococcal strains G148 and G43 and the group C streptococcal strain C40. The 65-kDa G148 protein G and the 58-kDa C40 protein G showed affinity for both immunoglobulin G (IgG) and human serum albumin (HSA), whereas the 40-kDa G43 protein G bound only IgG. Despite the different molecular patterns, the three protein G species had identical NH2-terminal amino acid sequences. Apart from the 65-kDa peptide, digestion of G148 streptococci with mutanolysin also produced a 52-kDa IgG- and HSA-binding peptide and a 14-kDa HSA-binding peptide. It was demonstrated that these peptides resulted from cleavage of 65-kDa protein G by proteolytic components in the mutanolysin preparation. The protein G genes of the C40 and G43 strains were cloned and sequenced, and their structure was compared to the previously published sequence of the G148 protein G gene. As compared to G148, both the C40 and G43 genes lacked a 210-base pair fragment in the IgG-binding region, accounting for the 10-fold lower affinity of these proteins for IgG. The G43 gene also lacked a 450-base pair fragment in the 5'-end of the gene, explaining why the G43 protein G did not bind HSA. The differences in protein G structure did not correlate with the clinical origin of the strains used in this study. The IgG-binding region of protein G was further mapped. Thus, a peptide corresponding to a single IgG-binding unit was obtained by the cloning and expression of a 303-base pair polymerase chain reaction-generated DNA fragment. The affinity of this 11.5-kDa peptide for human IgG was 8.0 x 10(7) M-1, as determined by Scatchard plots. Finally, a 55-amino acid-long synthetic peptide, corresponding to one of the three repeated domains in the COOH-terminal half of strain G148 protein G, effectively blocked binding of protein G to IgG.     

Proteome‐wide analysis of phospho‐regulated PDZ domain interactions

A key function of reversible protein phosphorylation is to regulate protein–protein interactions, many of which involve short linear motifs (3–12 amino acids). Motif‐based interactions are difficult to capture because of their often low‐to‐moderate affinities. Here, we describe phosphomimetic proteomic peptide‐phage display, a powerful method for simultaneously finding motif‐based interaction and pinpointing phosphorylation switches. We computationally designed an oligonucleotide library encoding human C‐terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. We incorporated these oligonucleotides into a phage library and screened the PDZ (PSD‐95/Dlg/ZO‐1) domains of Scribble and DLG1 for interactions potentially enabled or disabled by ligand phosphorylation. We identified known and novel binders and characterized selected interactions through microscale thermophoresis, isothermal titration calorimetry, and NMR. We uncover site‐specific phospho‐regulation of PDZ domain interactions, provide a structural framework for how PDZ domains accomplish phosphopeptide binding, and discuss ligand phosphorylation as a switching mechanism of PDZ domain interactions. The approach is readily scalable and can be used to explore the potential phospho‐regulation of motif‐based interactions on a large scale.     

A monovalent mutant of cyanovirin-N provides insight into the role of multiple interactions with gp120 for antiviral activity

Cyanovirin-N (CV-N) is a 101 amino acid cyanobacterial lectin with potent antiviral activity against HIV, mediated by high-affinity binding to branched N-linked oligomannosides on the viral surface envelope protein gp120. The protein contains two carbohydrate-binding domains, A and B, each of which binds short oligomannosides independently in vitro. The interaction to gp120 could involve either a single domain or both domains simultaneously; it is not clear which mode would elicit the antiviral activity. The model is complicated by the formation of a domain-swapped dimer form, in which part of each domain is exchanged between two monomers, which contains four functional carbohydrate-binding domains. To clarify whether multivalent interactions with gp120 are necessary for the antiviral activity, we engineered a novel mutant, P51G-m4-CVN, in which the binding site on domain A has been knocked out; in addition, a [P51G] mutation prevents the formation of domain-swapped dimers under physiological conditions. Here, we present the crystal structures at 1.8 A of the free and of the dimannose-bound forms of P51G-m4-CVN, revealing a monomeric structure in which only domain B is bound to dimannose. P51G-m4-CVN binds gp120 with an affinity almost 2 orders of magnitude lower than wt CV-N and is completely inactive against HIV. The tight binding to gp120 is recovered in the domain-swapped version of P51G-m4-CVN, prepared under extreme conditions. Our findings show that the presence of at least two oligomannoside-binding sites, either by the presence of intact domains A and B or by formation of domain-swapped dimers, is essential for activity.     

Chemical synthesis and evaluation of a backbone‐cyclized minimized 2‐helix Z‐domain

The Z‐molecule is a small, engineered IgG‐binding affinity protein derived from the immunoglobulin‐binding domain B of Staphylococcus aureus protein A. The Z‐domain consists of 58 amino acids forming a well‐defined antiparallel three‐helix structure. Two of the three helices are involved in ligand binding, whereas the third helix provides structural support to the three‐helix bundle. The small size and the stable three‐helix structure are two attractive properties comprised in the Z‐domain, but a further reduction in size of the protein is valuable for several reasons. Reduction in size facilitates synthetic production of any protein‐based molecule, which is beneficial from an economical viewpoint. In addition, a smaller protein is easier to manipulate through chemical modifications. By omitting the third stabilizing helix from the Z‐domain and joining the N‐ and C‐termini by a native peptide bond, the affinity protein obtains the advantageous properties of a smaller scaffold and in addition becomes resistant to exoproteases. We here demonstrate the synthesis and evaluation of a novel cyclic two‐helix Z‐domain. The molecule has retained affinity for its target protein, is resistant to heat treatment, and lacks both N‐ and C‐termini. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Design of stability at extreme alkaline pH in streptococcal protein G

Protein G (PrtG) is widely used as an affinity-based ligand for the purification of IgG. It would be desirable to improve the resistance of affinity chromatography ligands, such as PrtG, to commercial cleaning-in-place procedures using caustic alkali (0.5 M NaOH). It has been shown that Asn residues are the most susceptible at extreme alkaline pH: here, we show that replacement of all three Asn residues within the IgG-binding domain of PrtG only improves stability towards caustic alkali by about 8-fold. Study of the effects of increasing pH on PrtG by fluorescence and CD shows that the protein unfolds progressively between pH 11.5 and 13.0. Calculation of the variation in electrostatic free energy with pH indicated that deprotonation of Tyr, Lys and Arg side-chains at high pH would destabilize PrtG. Introduction of the triple mutation Y3F/T16I/T18I into PrtG stabilized it by an extra 6.8 kcal/mol and the unfolding of the protein occurred at a pH of about 13, or 1.5 pH units higher than wild type. The results show that strategies for the stabilization of proteins at extreme alkaline pH should consider thermodynamic stabilization that will retain the tertiary structure of the protein and modification of surface electrostatics, as well as mutation of alkali-susceptible residues.     

Common structural requirements for heptahelical domain function in class A and class C G protein-coupled receptors

G protein-coupled receptors (GPCRs) are key players in cell communication. Several classes of such receptors have been identified. Although all GPCRs possess a heptahelical domain directly activating G proteins, important structural and sequence differences within receptors from different classes suggested distinct activation mechanisms. Here we show that highly conserved charged residues likely involved in an interaction network between transmembrane domains (TM) 3 and 6 at the cytoplasmic side of class C GPCRs are critical for activation of the gamma-aminobutyric acid type B receptor. Indeed, the loss of function resulting from the mutation of the conserved lysine residue into aspartate or glutamate in the TM3 of gamma-aminobutyric acid type B(2) can be partly rescued by mutating the conserved acidic residue of TM6 into either lysine or arginine. In addition, mutation of the conserved lysine into an acidic residue leads to a nonfunctional receptor that displays a high agonist affinity. This is reminiscent of a similar ionic network that constitutes a lock stabilizing the inactive state of many class A rhodopsin-like GPCRs. These data reveal that despite their original structure, class C GPCRs share with class A receptors at least some common structural feature controlling G protein activation.     

A green approach toward antibody purification: a sustainable biomimetic ligand for direct immobilization on (bio)polymeric supports

This paper presents a sustainable strategy for improving the capture of antibodies by affinity chromatography. A novel biomimetic ligand (4‐((4‐chloro‐6‐(3‐hydroxyphenoxy)‐1,3,5‐triazin‐2‐yl)oxy)naphthalen‐1‐ol) (TPN‐BM) was synthesized using a greener and simple protocol to overcome solubility limitations associated with ligand 22/8, known as artificial protein A. Furthermore, its subsequent immobilization on chitosan‐based monoliths induced by plasma surface activation allowed the design of a fast and efficient chromatographic platform for immunoglobulin G (IgG) purification. The TPN‐BM functionalized monoliths exhibited high‐binding capacity (160 ± 10 mg IgG per gram of support), and a selective capture of monoclonal antibodies directly from mammalian crude extracts in 85 ± 5% yield and 98% of purity. The synthesis of ligand TPN‐BM and the routes followed for monoliths preparation and functionalization were inspired in the green chemistry principles allowing the reduction of processing time, solvents and purification steps involved, turning the integrated system attractive from an economical and chemical point of view. Copyright © 2013 John Wiley & Sons, Ltd.     

Molecular cloning and expression of the mouse high affinity Fc receptor for IgG

Full length cDNA clones encoding the mouse Fc gamma RI were isolated by using redundant oligonucleotide probes based on previously determined amino acid sequence of protein bound to an IgG2a antibody column. Sequence analysis of cDNA clones indicates that mouse Fc gamma RI is a transmembrane glycoprotein that is composed of three disulfide bonded extracellular Ig binding domains unlike Fc gamma RII of man and mouse. These extracellular domains contain five potential sites of N-linked glycosylation; three sites in the first domain and one in each of the second and third domains. In addition a transmembrane region is present followed by a cytoplasmic tail of 84 amino acids. Analysis of the amino acid sequence of the first two extracellular domains of Fc gamma RI indicate that these are highly homologous to the extracellular domains of Fc gamma RII; the third domain is different and shows a lower level of homology to other FcR domains but is clearly related to the Ig super-family. Transfected cells expressing Fc gamma RI were shown to bind immune complexes of rabbit IgG; and monomeric IgG2a bound to transiently transfected cells with an affinity of approximately 5 x 10(7) M-1, i.e. the receptor was of high affinity and therefore was by definition Fc gamma RI. Northern analysis demonstrated that Fc gamma RI mRNA could be detected in the Fc gamma RI+ myeloid cell lines WEH1 3B and J774. Finally, Southern analysis indicated that Fc gamma RI is likely to be encoded by a single copy gene of approximately 9 kb.