Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.17 (lysozyme)
 21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have isolated from a human synthetic phage display library a clone, 2A3, which discriminates native lysozyme from denatured forms. Binding of single-chain Fv fragments (scFvs) of the clone to native hen egg white lysozyme was competitively inhibited by native hen egg white (hew) and human (h) lysozymes. Dot blotting analysis indicated that scFv of the clone did not react with denatured lysozymes. The K(d) values for scFv of 2A3 binding to native hew- and h-lysozymes were 3.78 x 10(-9) and 9.31 x 10(-9) M, respectively, indicating that 2A3 binds more strongly to native hew-lysozyme than to native h-lysozyme. The deduced amino acid sequence of the V(H) chain-CDR3 region of 2A3 was RRYALDY, of which the Arg residues at positions 1 and 2 of the CDR3 region were observed to be extremely rare in other antibodies by homology analysis. Based on these observations, site-directed mutagenesis of the RRYALDY-coding region was carried out. The results, combined with biomolecular analyses, demonstrated that Arg residues at positions 1 and 2 of this region were important for native lysozyme-binding.
 ...
PMID:A single-chain Fv fragment 2A3 specific for native lysozyme: isolation from a human synthetic phage display antibody library and characterization. 1117 25

A recombinant strain of Aspergillus awamori expressing anti-lysozyme single chain antibody fragments (scFv), under the control of a xylanase promoter, was studied in order to investigate the impact of medium, induction regime and protease production on the expression of the product. Experiments with the time of induction showed that the optimum results are achieved when induction is started in the late exponential phase (21 h after inoculation) improving the titer of the product from 14.5 mg L(-1), obtained in the early exponential phase (7 h after inoculation), to 16.2 mg L(-1). A 100% increase of the carbon (fructose) and nitrogen (ammonium sulfate) sources in the growth medium resulted in an increase in product concentration from 16.2 to 108.9 mg L(-1) and an increase in maximum dry cell weight from 7.5 to 11.5 g L(-1). A 50% reduction in the concentration of the inducer resulted in an increase in the product yield from 10 mg g(-1) dry cell weight to 12 mg g(-1). Proteolytic enzymes were produced during the fermentation up to concentrations equivalent to 1.4 g L(-1) trypsin, but they had no detrimental effect on the concentration of the antibody fragment.
 ...
PMID:Factors affecting the production of a single-chain antibody fragment by Aspergillus awamori in a stirred tank reactor. 1148 20

The possibility of obtaining from any antibody a fluorescent conjugate which responds to the binding of the antigen by a variation of its fluorescence, would be of great interest in the analytical sciences and for the construction of protein chips. This possibility was explored with antibody mAbD1.3 directed against hen egg white lysozyme. Rules of design were developed to identify the residues of the antibody to which a fluorophore could be chemically coupled, after changing them to cysteine by mutagenesis. These rules were based on: the target residue belonging to a topological neighbourhood of the antigen in the structure of the complex between antibody and antigen; its absence of functional importance for the interaction with the antigen; and its solvent accessibility in the structure of the free antibody. Seventeen conjugates between the single-chain variable fragment scFv of mAbD1.3 and an environment-sensitive fluorophore were constructed. For six of the ten residues which fully satisfied the design rules, the relative variation of the fluorescence intensity between the free and bound states of the conjugate was comprised between 12 and 75% (in non-optimal buffer), and the affinity of the conjugate for lysozyme remained unchanged relative to the parental scFv. In contrast, such results were true for only one of the seven residues which failed to satisfy one of the rules and were used as controls. One of the conjugates was studied in more detail. Its fluorescence increased proportionally to the concentration of lysozyme in a nanomolar range, up to 90% in a defined buffer, and 40% in serum. This increase was specific for hen egg lysozyme and it was not observed with a closely related protein, turkey egg lysozyme. The residues which gave operational conjugates (six in V(L) and one in V(H)), were located in the immediate vicinity of residues which are functionally important, along the sequence of FvD1.3. The results suggest rules of design for constructing antigen-sensitive fluorescent conjugates from any antibody, in the absence of structural data.
 ...
PMID:Knowledge-based design of reagentless fluorescent biosensors from recombinant antibodies. 1205 49

Extracellularly expressed anti-hen egg lysozyme single-chain antibody fragments (scFv) produced by Aspergillus awamori were recovered using filtering centrifugation. Two filtering centrifuges with 0.5- and 30-L capacities were used to represent laboratory- and pilot-scale equipment, respectively. Critical regime analysis using the computational fluid dynamics (CFD) technique provided information about the local energy dissipation rates in both units. Experimental data indicated loss of scFv activity for energy dissipation rates above about 2.0 x 10(4) W kg(-1). This loss of activity increased in the presence of gas-liquid interfaces during filtering centrifugation. An ultra scaledown filtering centrifuge with a maximum working volume of 35 mL was designed to mimic the operating conditions identified by the critical regime analysis for the laboratory- and pilot-plant-scale units. The recovered scFv activity levels and the separation performance of the three units were comparable when operated at equal maximum energy dissipation rates.
 ...
PMID:Ultra scaledown to predict filtering centrifugation of secreted antibody fragments from fungal broth. 1211 1

Protein and other antigens typically have a number of different epitopes. This presents an opportunity for designing high-affinity antibodies by connecting via a flexible peptide linker two antibody fragments recognizing non-overlapping epitopes on the same antigen. The same strategy was employed in natural and designed DNA-binding proteins. According to a previous theory, the linking enhances the antigen-binding affinity over those of the individual antibody fragments (with association constants K(A) and K(B)) by p(d(0))K(B) or p(d(0))K(A), where p(d(0))=(3/4pil(p)bL)(3/2)exp(-3d(0)(2)/4l(p)bL)(1-5l(p)/4bL+ cdots, three dots, centered ) is the probability density for the end-to-end vector of the flexible linker with L residues to have a distance d(0). The predicted affinity enhancement is found to be actually approached by a bi-specific antibody against hen egg lysozyme consisting of scFv fragments of D1.3 and HyHEL-10. The wide applicability of the theory is demonstrated by diverse examples of protein-protein interactions constrained by flexible linkers.
 ...
PMID:Quantitative account of the enhanced affinity of two linked scFvs specific for different epitopes on the same antigen. 1274 13

Many attempts have been made to develop antibody fragments that can be expressed in the cytoplasm ("intrabodies") in a stable and functional form. The recombinant antibody fragment scFv(F8) is characterised by peculiarly high in vitro stability and functional folding in both prokaryotic and eukaryotic cytoplasm. To dissect the relative contribution of different scFv(F8) regions to cytoplasmic stability and specificity we designed and constructed five chimeric molecules (scFv-P1 to P5) in which several groups of residues important for antigen binding in the poorly stable anti-hen egg lysozyme (HEL) scFv(D1.3) were progressively grafted onto the scFv(F8) scaffold. All five chimeric scFvs were expressed in a soluble form in the periplasm and cytoplasm of Escherichia coli. All the periplasmic oxidised forms and the scFv(P3) extracted from the cytoplasm in reducing conditions had HEL binding affinities essentially identical (K(d)=15nM) to that of the cognate scFv(D1.3) fragment (K(d)=16nM). The successful grafting of the antigen binding properties of D1.3 onto the scFv(F8) opens the road to the exploitation of this molecule as a scaffold for the reshaping of intrabodies with desired specificities to be targeted to the cytoplasm.
 ...
PMID:Engineering stable cytoplasmic intrabodies with designed specificity. 1282 71

The fluorescence of tryptophan is used as a signal to monitor the unfolding of proteins, in particular the intensity of fluorescence and the wavelength of its maximum lambda(max). The law of the signal is linear with respect to the concentrations of the reactants for the intensity but not for lambda(max). Consequently, the stability of a protein and its variation upon mutation cannot be deduced directly from measurements made with lambda(max). Here, we established a rigorous law of the signal for lambda(max). We then compared the stability DeltaG(H(2)O) and coefficient of cooperativity m for a two-state equilibrium of unfolding, monitored with lambda(max), when the rigorous and empirical linear laws of the signal are applied. The corrective terms involve the curvature of the emission spectra at their lambda(max) and can be determined experimentally. The rigorous and empirical values of the cooperativity coefficient m are equal within the experimental error for this parameter. In contrast, the rigorous and empirical values of the stability DeltaG(H(2)O) generally differ. However, they are equal within the experimental error if the curvatures of the spectra for the native and unfolded states are identical. We validated this analysis experimentally using domain 3 of the envelope glycoprotein of the dengue virus and the single-chain variable fragment (scFv) of antibody mAbD1.3, directed against lysozyme.
 ...
PMID:Quantitative measurement of protein stability from unfolding equilibria monitored with the fluorescence maximum wavelength. 1608 53

Numerous approaches have been described to obtain variable fragments of antibodies (Fv or scFv) that are sufficiently stable for their applications. Here, we combined several knowledge-based methods to increase the stability of pre-existing scFvs by design. Firstly, the consensus sequence approach was used in a non-stringent way to predict a large basic set of potentially stabilizing mutations. These mutations were then prioritized by other methods of design, mainly the formation of additional hydrogen bonds, an increase in the hydrophilicity of solvent exposed residues, and previously described mutations in other antibodies. We validated this combined method with antibody mAbD1.3, directed against lysozyme. Fourteen potentially stabilizing mutations were designed and introduced into scFvD1.3 by site-directed mutagenesis, either individually or in combinations. We characterized the effects of the mutations on the thermodynamic stability of scFvD1.3 by experiments of unfolding with urea, monitored by spectrofluorometry, and tested the additivity of their effects by double-mutant cycles. We also quantified the individual contributions of the resistance to denaturation ([urea](1/2)) and cooperativity of unfolding (m) to the variations of stability and the energy of coupling between mutations by a novel approach. Most mutations (75%) were stabilizing and none was destabilizing. The progressive recombination of the mutations into the same molecule of scFvD1.3 showed that their effects were mostly additive or synergistic, provided a large overall increase in protein stability (9.1 kcal/mol), and resulted in a highly stable scFvD1.3 derivative. The mechanisms of the mutations and of their combinations involved variations in the resistance to denaturation, cooperativity of unfolding, and likely residual structures of the denatured state, which was constrained by two disulfide bonds. This combined method should be applicable to any recombinant antibody fragment, through a single step of mutagenesis.
 ...
PMID:Improving the stability of an antibody variable fragment by a combination of knowledge-based approaches: validation and mechanisms. 1692 23

Llamas possess a class of unconventional immunoglobulins that have only heavy chains; unpaired heavy variable domains are responsible for antigen binding. These domains have previously been cloned and expressed as single domain antibodies (sdAbs); they comprise the smallest known antigen binding fragments. SdAbs have been shown to bind antigens at >90 degrees C and to refold after being denatured. To take advantage of the remarkable properties of sdAbs, we constructed a large, semisynthetic llama sdAb library. This library facilitated the rapid selection of binders to an array of biothreat targets. We selected sdAb specific for live vaccinia virus (a smallpox virus surrogate), hen egg lysozyme, cholera toxin, ricin, and staphylococcal enterotoxin B. The selected sdAb possessed high specificity as well as enhanced thermal stability in comparison to conventional IgG and scFv antibodies. We also determined equilibrium dissociation constants as well as demonstrated the use of several antitoxin sdAbs as effective capture and reporter molecules in sandwich assays on the Luminex instrument. The ability to rapidly select such rugged antibodies will enhance the reliability of immunoassays by extending shelf life and the capacity to function in hostile environments.
 ...
PMID:Facile generation of heat-stable antiviral and antitoxin single domain antibodies from a semisynthetic llama library. 1716 13

High affinity and discriminating specificity are important parameters for any successful antibody based targeting strategy. We herein describe a system for the construction and subsequent selection of affinity-optimised chelating recombinant antibodies (CRAbs) from a randomised filamentous phage-display inter-scFv linker library. Using a simple, robust and highly degenerate tandem scFv cloning strategy a phage-display library of CRAbs with varied inter-scFv linkers was constructed and characterised. The library consisted of two single-chain Fvs (scFvs) of well characterised anti-lysozyme antibodies D1.3 and HyHEL-10(TF), specific for distinct non-overlapping epitopes, separated by flexible polypeptide linkers of varying lengths and sequences. The use of a stringent affinity-based selection strategy quickly led to the enrichment of CRAbs with a restricted set of linker lengths (16-21 amino acids) which agrees very closely with previously described crystal structure data, affinity measurements and mathematical modelling. This CRAb linker phage-display selection strategy is a widely applicable approach for the selection of very high affinity CRAbs for pairs of scFvs against potentially any target antigen, complementing the more arbitrary affinity maturation approaches based on random mutagenesis.
 ...
PMID:Phage display of chelating recombinant antibody libraries. 1735 51


<< Previous 1 2 3 Next >>