Chimeric IgG-binding Receptors Engineered from Staphylococcal Protein A and Streptococcal Protein G *

Chimeric Fc receptors, consisting of the IgG-binding domains of both staphylococcal protein A and streptococcal protein G, were constructed. An efficient bacterial expression system was used to produce the recombinant proteins, which vary in size and number of IgG-binding  domains.  The  purified  receptors were analyzed by immunodiffusion and a competitive enzyme-linked immunosorbent assay to establish the relative binding strength to various polyclonal and monoclonal immunoglobulins from different species. The results demonstrate that protein A and protein G have complementary binding patterns and that the chimeric receptors retain the binding capacities of both the parental constituents. This suggests that these novel chimeric receptors might be versatile reagents for immunochemical assays.

11 To whom correspondence should be sent.
The abbreviations used are: SPA, staphylococcal protein A; SPG, streptococcal protein G. ular those involving IgG with weak binding to SPA, such as sheep IgG, some mouse monoclonals, and human IgG3.
However, a detailed evaluation of the binding strength of SPG to various immunoglobulins was hampered due to the difficulties of dissolving the Fc receptor from the bacterial cell wall. Treatments using trypsin or pepsin (Myhre and Kronvall, 1981), specific phages (Christensen and Holm, 1976), acid (Havlicek, 1978), alkali (Grubb et al., 1982), and papain (Bjorck and Kronvall, 1984) gave heterogeneous materials with molecular weights ranging from 30,000 (Grubb et al., 1982) to 100,000 (Havlicek, 1978). This might explain the conflicting binding patterns of SPG reported. A method to obtain large quantities of a more well defined material is, therefore, desired.
Recently, the gene coding for protein G was isolated Fahnestock et al., 1986), which allows production of a recombinant protein. Using this approach,  compared the relative binding of SPA and SPG to different immunoglobulins at physiological conditions. The results suggested, in contrast to earlier reports of proteasereleased material, that protein G is not superior to protein A in its binding to all immunoglobulins but rather has a complementary binding pattern, i.e. protein G binds stronger than protein A to polyclonal IgG from cow, horse, and sheep, while the reverse was observed for polyclonal IgG from guinea pig and dog. This paper describes the production of a defined and homogeneous recombinant protein G and reports on its binding characteristics. The results indicate that it might be possible to obtain an optimal IgG-binding receptor by combining the binding activities of the class I and the class I11 receptors. We have, therefore, by gene fusion techniques constructed also two chimeric Fc receptors consisting of the IgG-binding domains of both SPA and SPG, varying in size and number of IgG-binding domains. Immunological assays demonstrate that these novel receptors retain the binding capacities of both SPA and SPG.

MATERIALS AND METHODS AND RESULTS AND DISCUSSION*
Functional Analysis of the Novel IgG-binding Receptors by Immunodiffusion-Different gene fragments encoding parts of staphylococcal protein A and streptococcal protein G were used to assemble the three constructs schematically outlined in Fig. 2, B, C, and D. These are compared to the structure of Empty boxes represent coding sequences not relevant for I g G binding.
A , protein A obtained from S. aureu; B, protein G as produced by the recombinant host; C, protein AG; and D, protein ZZG. commercially available protein A ( Fig. 2A). A coupled expression/secretion system in Escherichia coli  was used to obtain substantial amounts of each receptor.

TJINEA-PIG
To reveal the reactivity of the chimeric receptors to the Fc portion, immunodiffusion studies were performed using various IgG species and classes (Fig. 3). Polyclonal IgG from human, rat, cow, sheep, dog, and guinea pig serum were selected to represent immunoglobulins with differential binding to native protein A and G . Both proteins react with human IgG, while protein A in addition reacts with IgG from dog and guinea pig (Fig. 3A) and protein G with IgG from cow and sheep (Fig. 3B). This demonstrates that the truncated 23,000 protein G molecule exhibits the same binding pattern as the native protein G receptor described earlier . None of the receptors precipitated with rat polyclonal IgG.
The immunodiffusion assay using the chimeric receptors is presented in Fig. 3, C and D. The protein AG receptor precipitates with IgG from all six species except rat, while protein ZZG shows the same binding pattern, except that no precipitate with IgG from dog could be detected. Thus, both receptors have gained binding properties of both protein A and protein G, demonstrating that the novel receptors are also functionally chimeric. Comparative Binding to Polyclonal IgG-A competitive enzyme-linked immunosorbent assay was used to investigate the ability of native and recombinant Fc receptors to bind to polyclonal IgG from different species. The results presented in Table I demonstrate differences in the relative binding pattern between protein A and protein G. Protein A has a significantly higher affinity to polyclonal IgG from dog, pig, mouse, and guinea pig, while the opposite is true for IgG from cow, horse, and sheep. Protein A binds slightly better to human IgG, while protein G binds somewhat stronger to goat IgG. None of the receptors interacts with chicken IgG, and the binding to rat IgG is in both cases weak. The two proteins, therefore, exhibit complementary binding patterns for mammalian IgG. A similar pattern has been reported earlier for protein A and an Fc receptor from a group C streptococcus (Reis et al., 1984). Reis et al. (1986) reported on different binding patterns and concluded that the streptococcal class I11 Fc receptor reacts both stronger and with a wider range of IgG species than staphylococcal protein A. However, the differences in relative affinity between their results and the data presented here (Table I) are probably due to differences in assays, binding conditions, and origin of the protein G molecule. The remarkable difference in pH dependence of the binding of various IgG by protein G and protein A, noted by Akerstrom and Bjorck (1986)) emphasizes that direct comparisons of binding assays performed at different conditions might be hazardous.
The immunodiffusion studies of the chimeric receptors ( Fig.  3) indicated that the novel receptors consisting of both the class I and the class I11 receptors might have a combined binding capacity of the parental proteins. The results of the inhibition studies presented in Table I support this conclusion, although quantitative differences were observed for protein AG and protein ZZG. An interesting feature is that in all cases where protein A shows high affinity, the binding capacity of protein AG exceeds protein ZZG and vice versa. This probably reflects the fact that protein AG has five IgG-binding domains of protein A, while protein ZZG only contains two protein A-like domains.
The relative amount of the various immunoglobulins needed to inhibit the binding of rabbit IgG to the chimeric receptors is usually slightly higher than the values for the parents. The reduction in binding may depend on differences in molecular weight or the molar amounts of the relevant IgG-binding domains. The binding of the four receptors (A, G, AG, and ZZG) to the microtiter well may also be influenced by factors such as size and overall structure of the proteins. However, despite the semi-quantitative nature of the assay,  it is possible to conclude from Table I that the novel chimeric receptors AG and ZZG have a wider range of IgG species reactivity than either of the two parental protein A or protein G receptors. Comparative Binding to Human IgG Subclasses-The relative binding of the four receptors to human myeloma immunoglobulins i ! presented in Table 11. As shown earlier Akerstrom and Bjorck, 1986;Reis et al., 1986), protein G binds strongly to human IgG3 in contrast to protein A which shows low affinity to this subclass. Both the chimeric receptors bind IgG3, but the relative affinity is considerably lower than for protein G, especially for protein AG. It is possible that protein ZZG, which to 60% consists of protein G domains, has an IgG3-binding activity which is closer to that of protein G than protein AG with only 38% of protein G domains.
Table I1 also shows that proteins A and G have approximately the same binding activity to the other human subclasses, although protein A binds somewhat better to IgGl (X, K ) , IgG2 (X, K ) , and IgG4 (X). Since both parental proteins have similar affinities to subclasses 1, 2, and 4, identical binding of the chimeric receptors is also observed. Table 11, therefore, suggests that compared to protein G, the chimeric receptors exhibit no advantage in binding to human IgG subclasses. However, the chimeric receptors still maintain the ability to recognize all the human subclasses of IgG.
Comparative Binding to Mouse Monoclonal IgG-Although the binding pattern of the Fc receptor from streptococci to various immunoglobulins has been determined Reis et al., 1986), no quantitative study of the affinity t? mouse monoclonals of all subclasses has been reported. Akerstrom et al. (1985) determined the binding of three subclasses of radiolabeled mouse monoclonal antibodies to protein A and protein G coupled to Sepharose and nitrocellulose, respectively. They demonstrated higher affinity of two immunoglobulins of subclass 1 and one each of IgG2a and IgG3 to protein G as compared to protein A. It was later confirmed by Akerstrom and Bjorck (1986) that at least two of these monoclonals (of subclass IgGl and IgG2a) show higher binding to protein G than to protein A at pH between 4 and 8, while protein A binds equally well or stronger at higher pH.
Since subclass IgG2b was not included in these studies and only four monoclonals in total were used, we decided to determine the binding of protein A, protein G, and the two chimeric receptors to two independent monoclonal antibodies of each subclass. The results presented in Table I11 suggest that protein A has a relatively high affinity to IgG2a, while protein G binds stronger to IgG2b. The affinity to IgG3 is strong for both proteins but is weak to subc!ass IgG1. These results differ dramatically from those of Akerstrom et al. (1985), and the question arises if this is due to differences in binding conditions or individual variability of the monoclonal antibodies. It is interesting to note that the chimeric receptors (AG and ZZG) bind well to IgGSa, IgGZb, and IgG3. This further supports the conclusion that these novel receptors have gained the binding capacity of both parental receptors.
Concluding Remarks-A new concept to obtain novel receptors has been introduced in this paper. With the aid of gene fusions, the gene fragments encoding IgG-binding domains of staphylococcal protein A and streptococcal protein G were assembled, and the resulting proteins were shown to be structurally and functionally chimeric. The advantages of these novel chimeric receptors are several. First, the binding spectra toward polyclonal immunoglobulins from different species are broader than the individual parent proteins. Second, individual subclasses of mouse monoclonals are more likely to have strong affinity to the chimeras than to protein A or protein G itself. Third, the dramatic dependence on pH for binding to both protein A and G might be reduced. The fact that protein G has a low pH optimum (around pH 6), while the pH optimum for protein A is basic (pH 8), obviously enhances this positive effect. Finally, a multivalent receptor containing, in the case of protein AG, as many as eight IgG-binding domains might prove to enhance binding in certain applications, in which availability of the receptor is crucial. Nilsson et al. (1985) has earlier reported on a similar strategy to construct protein A-enzyme conjugates using genetic approaches. Gene fusions between protein A, @-galactosidase, or alkaline phosphatase were produced and shown to be functional both in IgG binding and enzymatic activity. This approach provided well defined and homogeneous in vivo coupled materials. Recently, such a @-galactosidase conjugate was used to detect specific antibodies in Western blots (Valerie et al., 1987). Using similar techniques an enzyme conjugate coupled to the protein AG molecule can be obtained. Such a tripartite molecule has recently been constructed and shown to be functional in immunoassay^.^ In conclusion, the results presented here demonstrate that gene fusion techniques are a very powerful tool to obtain novel proteins with improved functional properties. It is likely that chimeric receptors, such as the two described here, might in the near future prove to be useful for various immunological and biochemical applications.