Purification and some properties of streptococcal protein G, a novel IgG-binding reagent

Protein G, a bacterial cell wall protein with affinity for immunoglobulin G (IgG), has been isolated from a human group G streptococcal strain (G148). Bacterial surface proteins were solubilized by enzymatic digestion with papain. Protein G was isolated by sequential use of ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and affinity chromatography on Sepharose 4B-coupled IgG. The presence of protein G in various pools and fractions during the isolation was followed by their ability to inhibit the binding of radio-labeled IgG to G148 bacteria. A highly purified protein G was obtained. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the apparent m.w. was 30,000, and on agarose gel electrophoresis the purified protein gave rise to a single band in the alpha 1-region. Protein G was found to bind all human IgG subclasses and also rabbit, mouse, and goat IgG. On the IgG molecule, the Fc part appears mainly responsible for the interaction with protein G, although a low degree interaction was also recorded for Fab fragments. IgM, IgA, and IgD, however, showed no binding to protein G. This novel IgG-binding reagent promises to be of theoretical and practical interest in immunologic research.     

Fatty-acid-binding protein from bovine brain. Amino acid sequence and some properties

A fatty-acid-binding protein (FABP) from the cytosol of bovine brain was purified by Sephadex G-75 filtration and electrofocusing. The purified FABP behaved as an anionic protein with an apparent molecular mass of 14.7 kDa; its complete amino acid sequence was determined and microheterogeneity was observed. Sequence comparison with other FABPs of known sequence and the observed microheterogeneity demonstrated the presence in brain of several homologous FABPs closely related to heart FABP and bovine mammary-derived growth inhibitor (MDGI).     

Synthesis and mutagenesis of an IgG-binding protein based upon protein A of Staphylococcus aureus.

A novel protein able to bind with high affinity to the Fc fragment of IgG from a variety of animals has been produced by a gene synthesis approach. The IgG binding is accomplished by the presence of a single or two consecutive domains based upon domain B from protein A of Staphylococcus aureus. The IgG-binding moiety is fused to a peptide containing 21, 53 or 81 amino acids derived from the N-terminus of bovine DNase I. The latter is present to guide the expression of the protein in Escherichia coli into an inclusion body. This facilitates the high expression and recovery of the IgG-binding domains. The binding activity of this fusion protein is very close to that of the native protein A. Site-directed mutagenesis of the fusion protein and subsequent identification of changed binding interactions is reported.     

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.     

A synthetic IgG-binding domain based on staphylococcal protein A

A synthetic IgG-binding domain based on staphylococcal protein A was designed with the aid of sequence comparisons and computer graphic analysis. A strategy, utilizing non-palindromic restriction sites, was used to overcome the difficulties of introducing site-specific changes into the repetitive gene. A single mutagenized gene fragment was polymerized to different multiplicities, and the different gene products were expressed in Escherichia coli. Using this scheme, protein A-like proteins composed of different numbers of IgG-binding domains were produced. These domains were changed to lack asparagine--glycine dipeptide sequences as well as methionine residues and are thus, in contrast to native protein A, resistant to treatment with hydroxylamine and cyanogen bromide.     

High level expression of a synthetic gene coding for IgG-binding domain B of Staphylococcal protein A

A gene coding for one of the IgG-binding domains of Staphylococcal protein A, designated domain B, was chemically synthesized. This gene was tandemly repeated to give dimeric and tetrameric domain B genes by the use of two restriction enzymes which gave blunt ends. The genes were highly expressed in Escherichia coli to afford a large amount of dimeric and tetrameric domain B proteins. The single domain B protein was efficiently produced as a fusion protein with a salmon growth hormone fragment. The fusion protein was converted to monomeric domain B by cyanogen bromide cleavage. The CD spectra of the monomeric, dimeric and tetrameric domain B proteins were essentially the same as that of native form protein A, showing that their secondary structures were very similar. The dimeric and tetrameric domain B proteins formed precipitates with IgG as protein A. This system permits the efficient production of mutated single and multiple IgG-binding domains which can be used to study structural changes and protein A-immunoglobulin interactions.     

Synthesis and expression of an artificial gene of an IgG-binding fragment of protein A from Staphylococcus aureus

The chemical-enzymatic synthesis of a gene for IgG-binding fragment of the staphylococcal protein A has been carried out. The design of the gene, which consists of signal peptide and modified E and B domains, and strategy of the synthesis provided possibility of various degrees of polymerization of the gene fragment coding for B domain and of the whole gene. Several protein A-like polypeptides composed of the leader sequence, E domain and 1 to 4 copies of B domain were produced in E. coli cells under the lac promoter control.     

Sequential 1H NMR assignments and secondary structure of an IgG-binding domain from protein G

Protein G is a member of a class of cell surface bacterial proteins from Streptococcus that bind IgG with high affinity. A fragment of molecular mass 6988, which retains IgG-binding activity, has been generated by proteolytic digestion and analyzed by 1H NMR. Two-dimensional DQF-COSY, TOCSY, and NOESY spectra have been employed to assign the 1H NMR spectrum of the peptide. Elements of regular secondary structure have been identified by using nuclear Overhauser enhancement, coupling constant, and amide proton exchange data. The secondary structure consists of a central alpha-helix (Ala28-Val44), flanked by two portions of beta-sheet (Val5-Val26 and Asp45-Lys62). This is a fundamentally different arrangement of secondary structure from that of protein A, which is made up of three consecutive alpha-helices in free solution (Torigoe et al., 1990). We conclude that the molecular mechanisms underlying the association of protein A and protein G with IgG are different.     

Synthesis and properties of some novel anti-calmodulin drugs.

The preparation and properties of some novel inhibitors of calmodulin function are described. The compounds are cationic derivatives of phenyl-substituted thiazoles which inhibit the calmodulin stimulation of cyclic-AMP phosphodiesterase and are active against animal tumor cells in culture. These derivatives form the basis for the preparation of new, more potent inhibitors of calmodulin function which could take advantage of the reported elevated levels of calcium-bound calmodulin in tumor cells and show preferential anti-tumor activity.     

Function and structure of microvirid phage α3 genome. DNA sequence of H gene and properties of missense H mutant

The nucleotide sequence of wild-type α3 H gene and its surrounding region was determined and compared with those of ?X174 and G4. The corresponding DNA regions in double mutants amJH22, amJH69 and amJH76 were also sequenced and their missense mutation sites located. A phage strain missH22 having a single missense mutation in gene H was constructed by replacing the J region of amJH22 in vitro with the wild-type DNA. Like amJH22, the missense mutant coded for H protein with aberrant electrophoretic mobility, but formed normal plaques on suppressor-deficient Escherichia coli. Heat stability, plating efficiency on certain hosts and rate of eclipse were higher in strain missH22 than in wild-type phage.     

Function and structure of microvirid phage alpha 3 genome. DNA sequence of H gene and properties of missense H mutant

The nucleotide sequence of wild-type alpha 3 H gene and its surrounding region was determined and compared with those of phi X174 and G4. The corresponding DNA regions in double mutants amJH22, amJH69 and amJH76 were also sequenced and their missense mutation sites located. A phage strain missH22 having a single missense mutation in gene H was constructed by replacing the J region of amJH22 in vitro with the wild-type DNA. Like amJH22, the missense mutant coded for H protein with aberrant electrophoretic mobility, but formed normal plaques on suppressor-deficient Escherichia coli. Heat stability, plating efficiency on certain hosts and rate of eclipse were higher in strain missH22 than in wild-type phage.     

Structure of the IgG-binding regions of streptococcal protein G.

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.     

A novel Markov pairwise protein sequence alignment method for sequence comparison

The Smith-Waterman (SW) algorithm is a typical technique for local sequence alignment in computational biology. However, the SW algorithm does not consider the local behaviours of the amino acids, which may result in loss of some useful information. Inspired by the success of Markov Edit Distance (MED) method, this paper therefore proposes a novel Markov pairwise protein sequence alignment (MPPSA) method that takes the local context dependencies into consideration. The numerical results have shown its superiority to the SW for pairwise protein sequence comparison.