The gene encoding the IgG‐binding protein G from Streptococcus G148 was isolated by molecular cloning. A subclone containing a 1.5‐kb insert gave a functional product in Escherichia coli. Protein analysis of affinity‐purified polypeptides revealed two gene products, both smaller than protein G spontaneously released from streptococci, but with identical IgG‐binding properties. The complete nucleotide sequence of the insert revealed a repeated structure probably evolved through duplications of fragments of different sizes. The deduced amino acid sequence revealed an open reading frame extending throughout the insert, terminating in a TAA stop codon. Analysis of the two gene products by N‐terminal amino acid determination suggests that two different TTG codons are recognized in E. coli for initiation of translation to yield the two products. Based on these results several truncated gene constructions were expressed and analysed. The results suggest that the C‐terminal part of streptococcal protein G consists of three IgG‐binding domains followed by a region which anchors the protein to the cell surface. Structural and functional comparisons with streptococcal M protein and staphylococcal protein A have been made.

Streptococcal protein G is an IgG-binding receptor with a molecular weight of 63 kDa as predicted from the sequence of the corresponding gene. Here we show that a truncated recombinant protein of 23 kDa still has IgG-binding capacity and also interacts specifically with human serum albumin (HSA). This demonstrates that protein G is a bifunctional receptor. To investigate the structures needed for IgG- and albumin-binding, different parts of the receptor molecule were produced in E. coli using a coupled expression/secretion system. Affinity chromatography, using IgG or HSA immobilized on Sepharose, showed that the two binding activities are structurally separated. From these experiments, it was concluded that a region of 64 amino acid residues is sufficient for albumin-binding. The structure of this part of the protein suggests either a divalent or a trivalent binding capacity. The specific interaction to albumin was used to purify a heterologous protein by affinity chromatography to yield a pure fusion protein in a one-step procedure. The implication of this novel affinity system as a tool to facilitate protein immobilization and purification is discussed.

The gene encoding subtilisin Carlsberg from Bacillus licheniformis has been isolated by molecular cloning using a mixture of synthetic oligonucleotides. The entire nucleotide sequence of the coding sequence as well as 5' and 3' flanking sequences have been determined. The deduced amino acid sequence reveals an N-terminal signal peptide consisting of 29 residues, a pro-peptide of 76 residues followed by the mature protein comprising 274 residues. The ATG initiator codon is preceded by two putative overlapping ribosomal binding sequences. A palindromic sequence typical for transcription termination is found downstream from the TAA stop codon. Structural comparisons between different known subtilisin genes reveal extensive homology, particularly in the parts coding for the pro-region and the mature protein. Expression studies in Bacillus subtilis show that the cloned fragment produces a functional enzyme when inserted after a B. subtilis promoter.

The complete sequence of the structural gene encoding the immunoglobulin G binding protein from Streptococcus G148 has been determined, as well as its 5' and 3' flanking sequences. The sequence reveals an open reading frame encoding a putative preprotein with a relative molecular mass of 63294. N-Terminal sequencing of the mature protein, spontaneously released from streptococcal cells, demonstrates that the signal peptide consists of 33 amino acids. The DNA sequence reveals extensive internal homologies similar to other cell-wall-bound receptors from gram-positive bacteria. Comparisons with a related gene previously isolated from another strain of streptococci revealed large differences in size, due to variations in the number of internal repeats. The structure of the gene suggests an evolution through multiple duplications.Several gram-positive bacteria contain cell-surface proteins, probably involved in the pathogenicity of these bacteria in animals and man [l]. The best known receptor is the protein A from Staphylococcus aureus, which has found extensive use in quantitative and qualitative immunological techniques due to its binding to the Fc fragment of immunoglobulins [2]. Other receptors include the M protein from Streptococcus pyogenes [3], which is a virulence factor enabling the streptococci to resist clearance by phagocytic cells, and the fibronectin receptor from S. aureus which binds to fibronectin [4]. In addition, a number of immunoglobulin Fc receptors with differential binding to various IgG subclasses have been isolated from Streptococcus group A, C and G [5 -81.Recently, the genes encoding staphylococcal protein A [9] and streptococcal M protein [3] were cloned and sequenced. This revealed in both cases repetitive structures, suggesting an evolution of these genes through multiple duplications of internal fragments of different sizes. Both genes encode proteins consisting of a signal peptide followed by a repetitive receptor region, a proline-rich region and a hydrophobic C-terminus. Thus, although only low homology could be detected at the deduced primary sequence level, the overall structure of both genes suggests a common mechanism by which these receptors associate to the cell surface.We have earlier reported on the cloning of the IgG receptor from Streptococcus G148, called protein G, and the characterization of the part responsible for IgG binding [lo]. Here, we describe the cloning and sequencing of its 5'-end of the protein G gene from strain G148, as well as that of the 5' and 3' flanking regions. This has enabled structural comparisons to the related protein G gene from strain GX7809 [ll], showing large differences in size and number of internal repeats, despite high sequence homology. The results suggest a model for the evolution of this cell-wall-bound repector .

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