Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose.

The bacteria cell envelope is a complex multilayered structure that serves to protect these organisms from their unpredictable and often hostile environment. The cell envelopes of most bacteria fall into one of two major groups. Gram-negative bacteria are surrounded by a thin peptidoglycan cell wall, which itself is surrounded byan outer membrane containing lipopolysaccharide. Gram-positive bacteria lack an outer membrane but are surrounded by layers of peptidoglycan many times thicker than is found in the Gram-negatives. Threading through these layers of peptidoglycan are long anionic polymers, called teichoic acids. The composition and organization of these envelope layers and recent insights into the mechanisms of cell envelope assembly are discussed.

/pdf/the-bacterial-cell-envelope-o3k9u4ycci.pdf

The Bacterial Cell Envelope

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins.

Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

Polypeptides that are cleared from the kidney and do not contain in their original form a Fc region of an IgG are altered so as to comprise a salvage receptor binding epitope of an Fc region of an IgG and thereby have increased circulatory half-life.

Altered polypeptides with increased half-life

Compositions of, genetic constructions coding for, and methods for producing multivalent antigen-binding proteins are described and claimed. The methods include purification of compositions containing both monomeric and multivalent forms of single polypeptide chain molecules, and production of multivalent proteins from purified monomers. Production of multivalent proteins may occur by a concentration-dependent association of monomeric proteins, or by rearrangement of regions involving dissociation followed by reassociation of different regions. Bivalent proteins, including homobivalent and heterobivalent proteins, are made in the present invention. Genetic sequences coding for bivalent single-chain antigen-binding proteins are disclosed. Uses include all those appropriate for monoclonal and polyclonal antibodies and fragments thereof, including use as a bispecific antigen-binding molecule.

Multivalent antigen-binding proteins

Protein A was isolated from S. aureus and shown to precipitate about 45% of a pooled normal human γG preparation. In addition it was demonstrated that protein A reacts with myeloma γG-globulin, H chains from both normal and myeloma γG-globulins and the Fc fragment of normal γG-globulin. Thus, the reaction is not a true antigen-antibody reaction.

"Protein A" from S. aureus. I. Pseudo-immune reaction with human gamma-globulin.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2872%2980680-X

Protein a from Staphylococcus aureus. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins

Binding of immunoglobulins to protein A and immunoglobulin levels in mammalian sera.

A genetic approach is described to clarify the IgG-binding properties of the N-terminal portion of staphylococcal protein A (region E). Several gene fragments, encoding region E or B or protein A, have been cloned and expressed in Escherichia coli. The gene products were purified by IgG-affinity chromatography and subjected to structural and functional analyses. Both fragments can be efficiently purified using this method, suggesting that region B as well as region E has Fc-binding activity. In addition, gene fusions were assembled giving fragments EB and EE, which both showed a divalent Fc-binding. These results demonstrate that protein A consists of five IgG-binding domains. The implications of these findings for the structure of protein-A--immunoglobulin-G complexes are discussed.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1986.tb09625.x

Staphylococcal protein A consists of five IgG-binding domains.

Previous methods which have been used for the preparation of protein A, such as thermal release of protein A with lysozyme from the bacteria, have given a heterogenous product in a low yield. The present publication reports the isolation procedure of protein A from Staphylococcus aureus digested by lysostaphin for 2 h at 37°C. 450—500 mg of protein A was obtained from 300 g of wet bacteria. The preparation is homogenous as seen from its disc electrophoretic pattern.



Amino acid composition was determined and based on a molecular weight of 42000. The number of amino acid residues found was 378 ± 6. Less than 0.2% hexosamines was present. The ultraviolet spectrum and the absorption coefficient, A1%275nm= 1.65 of protein A is given. Less than 0.1 mol amino terminal amino acid per mol protein was detected which indicates that the protein has a blocked N-terminal amino acid. The protein was slowly digested with a mixture of carboxypeptidase A and B and 2 mol lysine/mol protein was released.



Precipitation of protein A with normal human γ-globulin gave an IgG: protein A molar ratio of 2.1:1 within the equivalence zone.



The results are discussed with regard to the subunit structure of the protein and the type of attachment of protein A to the peptidoglycan part of the cell wall.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1972.tb02023.x

John Sjöquist

Papers

"Protein A" from S. aureus. I. Pseudo-immune reaction with human gamma-globulin.

Protein a from Staphylococcus aureus. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins

Binding of immunoglobulins to protein A and immunoglobulin levels in mammalian sera.

Staphylococcal protein A consists of five IgG-binding domains.

Protein A Isolated from Staphylococcus aureus after Digestion with Lysostaphin