Preliminary crystal structure analysis of an Fab specific for a Salmonella O-polysaccharide antigen.

The Fab from an IgG1, lambda murine monoclonal antibody with specificity for the O-polysaccharide antigen of Salmonella typhimurium has been crystallized in the absence and presence of hapten. The conditions for crystal growth were vapor diffusion equilibration with 16 to 23% polyethylene glycol 8000 solutions. Data have been collected from crystals of the complex in space group P212121, a = 60.6 A, b = 111.3 A, c = 61.1 A, and refinement of a molecular replacement solution is underway.

Crystal structure to 2.45 A resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen.

The atomic structure of an antibody antigen-binding fragment (Fab) at 2.45 A resolution shows that polysaccharide antigen conformation and Fab structure dictated by combinatorial diversity and domain association are responsible for the fine specificity of the Brucella-specific antibody, YsT9.1. It discriminates the Brucella abortus A antigen from the nearly identical Brucella melitensis M antigen by forming a groove-type binding site, lined with tyrosine residues, that accommodates the rodlike A antigen but excludes the kinked structure of the M antigen, as envisioned by a model of the antigen built into the combining site. The variable-heavy (VH) and variable-light (VL) domains are derived from genes closely related to two used in previously solved structures, M603 and R19.9, respectively. These genes combine in YsT9.1 to form an antibody of totally different specificity. Comparison of this X-ray structure with a previously built model of the YsT9.1 combining site based on these homologies highlights the importance of VL:VH association as a determinant of specificity and suggests that small changes at the VL:VH interface, unanticipated in modeling, may cause significant modulation of binding-site properties.

Some Properties of Extracellular Acetylxylan Esterase Produced by the Yeast Rhodotorula mucilaginosa.

The red yeast Rhodotorula mucilaginosa produced an esterase that accumulated in the culture supernatant on induction with triacetin. The enzyme was specific for substrates bearing an O-acetyl group, but was relatively nonspecific for the rest of the molecule, which could consist of a phenol, a monosaccharide, a polysaccharide, or an aliphatic alcohol. The esterase was more active against acetylxylan and glucose beta-d-pentaacetate than were a number of esterases from plant and animal sources, when activities on 4-nitrophenyl acetate were compared. The enzyme exhibited Michaelis-Menten kinetics and was active over a broad pH range (5.5 to 9.2), with an optimum between pH 8 and 10. In addition, the enzyme retained its activity for 2 h at 55 degrees C. The yeast that produced the enzyme did not produce xylanase and, hence, is of interest for the production of acetylxylan esterase that is free of xylanolytic activity.

Production, crystallization and diffraction to atomic resolution of an antibody Fv specific for the blood-group A oligosaccharide antigen.

The histoblood-group ABO carbohydrate antigens are well known as important factors in blood transfusions, but they can also act as receptors for infectious agents and have been implicated in susceptibility to certain carcinomas. A single-chain variable-domain antigen-binding fragment (scFv) gene based on the known sequence of an anti-blood-group-A monoclonal antibody (AC1001) has been synthesized and expressed in Escherichia coli. The purified scFv preparation existed primarily in the monomeric form but also contained large amounts of dimeric and higher oligomeric forms. The corresponding variable-domain antigen-binding fragment (Fv) was generated by cleaving the VL-VH linker with subtilisin, and its activity was demonstrated by surface plasmon resonance with an immobilized bovine serum albumin-A-trisaccharide conjugate (KD = 290 microM). AC1001 Fv crystals grown in the presence of N-acetylgalactosamine diffracted to 0.93 A resolution. This is the first reported example of a crystal of an antibody antigen-binding fragment diffracting to atomic resolution.