To generate a stable resource from which high affinity human antibodies to any given antigen can be rapidly isolated, functional V-gene segments from 43 non-immunized human donors were used to construct a repertoire of 1.4 x 10(10) single-chain Fv (scFv) fragments displayed on the surface of phage. Fragments were cloned in a phagemid vector, enabling both phage displayed and soluble scFv to be produced without subcloning. A hexahistidine tag has been incorporated to allow rapid purification of scFv by nickel chelate chromatography. This library format reduces the time needed to isolate monoclonal antibody fragments to under two weeks. All of the measured binding affinities show a Kd < 10 nM and off-rates of 10(-3) to 10(-4) s-1, properties usually associated with antibodies from a secondary immune response. The best of these scFvs, an anti-fluorescein antibody (0.3 nM) and an antibody directed against the hapten DTPA (0.8 nM), are the first antibodies with subnanomolar binding affinities to be isolated from a naive library. Antibodies to doxorubicin, which is both immunosuppressive and toxic, as well as a high affinity and high specificity antibody to the steroid hormone oestradiol have been isolated. This work shows that conventional hybridoma technology may be superseded by large phage libraries that are proving to be a stable and reliable source of specific, high affinity human monoclonal antibodies.

Receptor-associated protein (RAP) was originally described as a 39-kDa intracellular protein copurifying with mammalian low density lipoprotein (LDL) receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2MR). RAP has a high affinity for LRP/alpha 2MR and interferes with the receptor's ability to bind a variety of ligands. The laying hen expresses, in a tissue-specific manner, at least four different proteins which belong to the same family of receptors as LRP/alpha 2MR. Here we show that the chicken also produces RAP, so far thought to be expressed only in mammals. Studies on the interaction of recombinant human RAP with the LDL receptor family in the chicken revealed that RAP binds with high affinity to the abundant oocyte receptor for yolk precursors (OVR) as well as to the somatic cell-specific LRP/alpha 2MR. Significantly, RAP interacts with a lower affinity with the LDL receptor, but does not bind to the oocyte-specific form of LRP. Binding of RAP to OVR inhibits the interaction of the receptor with all known physiological ligands, i.e. the yolk precursors very low density lipoprotein, vitellogenin, and alpha 2-macroglobulin. In COS cells transfected with OVR, RAP is internalized and degraded in a concentration-dependent and saturable manner. Lactoferrin, another protein with a high affinity for mammalian LRP/alpha 2MR, also binds to OVR and abolishes its interaction with yolk precursors. Cross-competition experiments show that RAP and lactoferrin recognize sites different from those involved in yolk precursor binding. The availability of pure OVR and LDLR enable us to determine kinetic parameters for the binding of RAP and lactoferrin to these receptors by surface plasmon resonance. Taken together, our results strongly suggest that chicken OVR, which is easily accessible and highly abundant in growing oocytes, represents a superior system for studying mechanistic and structural aspects of the interaction of ligands and modulating proteins with members of the LDL receptor gene family.

Trichoderma reesei was studied for its ability to produce fl-mannanase activity on a variety of carbon sources. The highest fl-mannanase activity was produced on cellulose, whereas fl-mannan-containing carbon sources (such as konjac powder or locust bean gum) gave lower enzyme titres. The enzyme responsible for the major fl-mannanolytic activity from T. reesei was purified to physical homogeneity by preparative chromatofocusing and anion exchange fast protein liquid chromatography. This fl-mannanase is a glycoprotein, with a molecular mass of 46 (+ 2) kDa and an isoelectric point of 5.2. It has an optimal pH at 5.0 and broad pH stability (2.5-7.0). It is stable for 60 min at 55 ° C, and has an optimal temperature for activity at 75 ° C. During incubation with locust bean gum, the enzyme releases mainly tri-and disaccharides.

Recently antibodies with a wide range of binding specificities have been isolated from large repertoires of antibody fragments displayed on filamentous phage, including those that are difficult to raise by immunization. We have used this approach to isolate an antibody fragment against chicken very low density lipoprotein (VLDL) receptor. It binds to the receptor with good affinity (K aff ‫؍‬ 2 ؋ 10 8 M ؊1) as measured by plasmon surface resonance, and competes for binding of natural ligands (vitellogenin, VLDL, and receptor-associated protein). The antibody also binds to other members of the low density lipoprotein (LDL) receptor family including rat LDL receptor and human and rat low density lipoprotein receptor-related protein (LRP/␣ 2 MR), and it competes for binding of receptor-associated protein to LRP/␣ 2 MR. Moreover, the antibody fragment inhibits infection of human fibroblasts deficient in LDL-R but expressing LRP/␣ 2 MR by human rhinovirus. Binding of the antibody is abolished upon reduction of the receptors and is strictly Ca 2؉ dependent. The phage antibody thus recognizes the ligand binding site(s) of several members of the LDL receptor family, in contrast to antibodies produced by hybridoma technology.The low density lipoprotein (LDL) 1 receptor of mammals is the prototype of a family of related proteins. Members of the LDL receptor family have several structural modules in common; (i) "binding repeats," complement-type domains consisting of ϳ40 residues displaying a triple disulfide bondstabilized negatively charged surface (head-to-tail combinations of these repeats are believed to specify ligand interaction); (ii) epidermal growth factor precursor-type repeats, also containing six cysteines each; (iii) modules of ϳ50 residues with a consensus tetrapeptide, Tyr-Trp-Thr-Asp (YWTD); and (iv), in the cytoplasmic region, signals for receptor internalization via coated pits, containing the consensus tetrapeptide Asn-Pro-Xaa-Tyr (NPXY).The LDL receptor family includes at least 4 proteins; the LDL receptor (LDL-R), the low density lipoprotein receptor related protein (also termed LRP/␣ 2 MR), gp330 (also termed megalin), and the very low density lipoprotein (VLDL) receptor. The LDL receptor (LDL-R) has a cluster of 7 binding repeats and binds to apolipoprotein B (apoB) and apolipoprotein E (apoE) (1, 2). LRP/␣ 2 MR is a giant receptor (4525 amino acids) containing 4 clusters of 2 to 11 binding repeats and has many ligands including apoE (3), ␣ 2 M-proteinase complexes (4, 5) among others (4 -12), and a 39-kDa intracellular protein (receptor-associated protein or RAP). RAP binds to LRP/␣ 2 MR with high affinity, co-purifies with LRP/␣ 2 M from liver and placenta (13,14), and competes for binding with all known LRP/␣ 2 MR ligands (4, 7-9, 15, 16). Whereas the majority of the ligands bound by LRP/␣ 2 MR fail to be recognized by LDL-R, human rhinoviruses (HRVs) of the minor receptor group type attach to either of these proteins (8). Recently, it was shown that RAP also binds to LDL-R but with much lower ...

Trichoderma reesei RUT C-30 formed an extracellular a-galactosidase when it was grown in a batch culture containing lactose or locust bean gum as a carbon source. Short-chain a-galactosides (melibiose, raffinose, stachyose), as well as the monosaccharides galactose, dulcitol, arabinose, and arabitol, also induced at-galactosidase activity both when they were used as carbon sources (at a concentration of 1%) in batch cultures and in resting mycelia (at concentrations in the millimolar range). The addition of 50 mM glucose did not affect the induction of ac-galactosidase formation by galactose. a-Galactosidase from T. reesei RUT C-30 was purified to homogeneity from culture fluids of galactose-induced mycelia. The active enzyme was a 50 ± 3-kDa, nonglycosylated monomer which had an isoelectric point of 5.2. It was active against several a-galactosides (p-nitrophenyl-t-D-galactoside, melibiose, raffinose, and stachyose) and galactomannan (locust bean gum) and was inhibited by the product galactose. It released galactose from locust bean gum and exhibited synergism with T. reesei 1-mannanase. Its activity was optimal at pH 4, and it displayed broad pH stability (pH 4 to 8). Its temperature stability was moderate (60 min at 50°C resulted in recovery of 70%o of activity), and its highest level of activity occurred at 60°C. Its action on galactomannan was increased by the presence of 13-mannanase. Hemicelluloses are the second most abundant polysaccharides in nature, and they seem to be linked to lignin in wood (23). The major constituents of hemicellulose are the hetero-1,4-0-D-xylans and the hetero-1,4-,B-D-mannans (galactoglucomannans and glucomannans). While heteroxylans are found mainly in grasses, cereals, and hardwoods, P-mannans are more abundant in softwoods (8, 23). The biotechnological interest in the hydrolysis of hemicelluloses for the pulp and paper industry and the feedstock industry has recently revived interest in the enzymology of hemicellulose degradation. In the case of galactomannans, this enzymatic hydrolysis requires the concerted action of the following hydrolytic enzymes: endo-1

Receptor-associated-protein (RAP) 1 was initially described as a 39 -40-kDa protein copurifying with LDL receptor (LDLR)-related protein (LRP) (1, 2). At the same time, a putative target antigen in Heymann nephritis (HN), an experimental rat model of human membranous nephropathy, was identified and cloned (3). On molecular characterization of RAP, it became evident that the protein identified as the target antigen in HN was identical to RAP and not, as previously assumed, a fragment of glycoprotein 330/megalin (4). RAP subsequently was shown to bind not only to LRP but also to other members of the LDLR family, including megalin (5), the VLDLR (6), the chicken oocyte receptor for VLDL and vitellogenin (LR8) (7,8) and, to a lesser extent, to the LDLR itself (8, 9). The ability to interfere with ligand binding to these receptors has made RAP a perfect tool to study ligand-receptor interaction both in vitro (8, 10 -14) and in vivo (15). The latter experiments especially have established the role of LRP as a chylomicron remnant receptor in the liver. Despite the successful use of RAP to study the physiological function(s) of members of the LDLR family and of LRP in particular, clues about the function of RAP in vivo emerged only recently. The tetrapeptide sequence HNEL at the carboxyl terminus of RAP was shown to be necessary for the retention of RAP in the endoplasmic reticulum; thus, RAP associates in vivo with LRP early in the secretory pathway and dissociates from the receptor before reaching the cell surface (16). These results suggested a specialized role of RAP as a chaperon for LRP, possibly regulating the interaction of the receptor with ligands along the secretory pathway. This was confirmed by elegant studies by Herz and colleagues in RAP knockout mice (17) and in cultured cells in experiments relating the biosynthesis and functional expression of LRP and megalin with that of RAP (18). These experiments show that at least one of the physiological functions of RAP can be defined as that of a specialized escort protein, which protects certain receptors from ligand-induced aggregation along their intracellular itinerary. We have previously shown that LR8 can serve as a model system to study ligand binding and structural aspects of LDLR family members (8,19,20). Here we have used LR8 to demonstrate that all receptors that strongly bind RAP share a common immunological epitope with the escort protein. Based on this observation, we propose a model for the development of passive Heymann nephritis induced by anti-RAP antibodies, in which megalin present in the brush borders of the proximal kidney tubule in rats constitutes an additional target antigen. EXPERIMENTAL PROCEDURESPreparation and Radiolabeling of Ligands-VLDL was prepared from plasma of estrogen-treated roosters by sequential ultracentrifugation according to the method of George et al. (21). VLDL was labeled with 125 I to a specific activity of 250 -400 cpm/ng using the iodine

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