Biomedical subjects

W Mandecki

Publications and source records attributed to W Mandecki.

At least 19 recordsLinked to original sources

PubMed1998

Unexpected frameshifts from gene to expressed protein in a phage-displayed peptide library.

A library of long peptides displayed on the pIII protein of filamentous phage was used in biopanning experiments against several protein targets. We find that a large percentage of phage clones that bind specifically to a target contain peptide-encoding genes that do not have an ORF. Instead, the reading frame is either interrupted by one or more nonsuppressed stop codons, or a post-transcriptional frameshift is needed to account for the expression of the minor phage coat protein pIII. The percentage of frameshifted clones varies depending on the target. It can be as high as 90% for clones specific for soluble forms of certain cytokine receptors. Conversely, biopanning against four mAbs did not yield any frameshifted clones. Our studies focused on one clone that binds specifically to rat growth hormone binding protein (GHBP) yet does not have an ORF. A secondary peptide library containing random mutations of this sequence was constructed and panned against GHBP to optimize and correct the reading frame. In the last round (round two) of panning with this library, none of the phage clones that bound to GHBP had an ORF. However, careful analysis of these clones allowed us to design a synthetic peptide capable of binding to GHBP. The results of this study indicate that ORFs are not required to obtain gene expression of the minor coat protein of filamentous phage and suggest that some ORF- clones may have a selective advantage over the clones having ORFs.

Amino Acid Sequence↗

PubMed1998

Identification of an allosteric binding site on the transcription factor p53 using a phage-displayed peptide library.

Monoclonal antibody PAb1620 recognizes a conformational epitope on the transcription factor p53 and, upon binding, allosterically inhibits p53 binding to DNA. A highly diverse (1.5 x 10(10) members) phage-displayed library of peptides containing 40 random amino acids was used to identify the PAb1620 binding site on p53. Panning this library against PAb1620 resulted in three unique peptides which have statistically significant sequence identities with p53 sufficient to identify the binding site as being composed of amino acids 106-113 and 146-156. Based on these results, we propose a mechanism by which PAb1620 can allosterically inhibit p53 binding to DNA through an indirect interaction between the antibody binding site and the L1 loop (amino acids 112-124) of p53, which is a component of the DNA binding region.

Allosteric Regulation↗

PubMed1998

Recombinant mouse-human chimeric antibodies as calibrators in immunoassays that measure antibodies to Toxoplasma gondii.

In the present study, we examined the feasibility of using recombinant antibodies containing murine variable regions and human constant regions as calibrators or controls in immunoassays. As a model system, we chose the Abbott IMx Toxo immunoglobulin M (IgM) and Toxo IgG assays designed to detect antibodies to Toxoplasma gondii. Two mouse monoclonal antibodies were selected based on their reactivity to the T. gondii antigens P30 and P66. Heavy- and light-chain variable-region genes were cloned from both hybridomas and transferred into immunoglobulin expression vectors containing human kappa and IgG1 or IgM constant regions. The constructs were stably transfected into Sp2/0-Ag14 cells. In the IMx Toxo IgG assay, immunoreactivity of the anti-P30 chimeric IgG1 antibody paralleled that of the positive human plasma-derived assay calibrators. Signal generated with the anti-P66 chimeric IgG1 antibody was observed to plateau below the maximal reactivity observed for the assay calibrator. Examination of the IgM chimeric antibodies in the IMx Toxo IgM assay revealed that both the anti-P30 and anti-P66 antibodies matched the assay index calibrator manufactured with human Toxo IgM-positive plasma. When evaluated with patient samples, the correlation between results obtained with the chimeric antibody calibrators and the positive human plasma calibrators was > or =0.985. These data demonstrate that chimeric mouse-human antibodies are a viable alternative to high-titer positive human plasma for the manufacture of calibrators and controls for diagnostic assays.

Amino Acid Sequence↗

PubMed1996

Discontinuous epitopes of hepatitis B surface antigen derived from a filamentous phage peptide library.

The structure of the small hepatitis B virus surface antigen (HBsAg) was investigated by epitope mapping of four anti-HBsAg monoclonal antibodies (mAbs). Amino acid sequences of epitopes were derived from affinity-enrichment experiments (biopanning) using a filamentous phage peptide library. The library consists of 10(9) different clones bearing a 30-residue peptide fused to gene III. Sequence homologies between peptides obtained from panning the library against the antibodies and the native HBsAg sequence allowed for precise description of the binding regions. Three of four mAbs were found to bind to distinct discontinuous epitopes between amino acid residues 101 and 207 of HBsAg. The fourth mAb was demonstrated to bind to residues 121-124. The sequence data are supported by ELISA assays demonstrating the binding of the HBsAg-specific peptides on filamentous phage to mAbs. The sequence data were used to map the surface of HBsAg and to derive a topological model for the alpha-carbon trace of the 101-207 region of HBsAg. The approach should be useful for other proteins for which the crystal structure is not available but a representative set of mAbs can be obtained.

Amino Acid Sequence↗

PubMed1996

In vitro selection of a high affinity antibody to oestradiol using a phage display human antibody library.

We have isolated a monoclonal antibody binding to oestradiol with high affinity (3.7 nM), and exhibiting a better than 1000-fold selectivity in binding to other steroids. A high affinity antibody with good specificity is essential for the accurate determination of circulating oestradiol levels. To date, conventional hybridoma technology has not yielded a reagent of sufficiently high affinity and specificity for this ligand. The aim of this study was to investigate whether such a reagent was accessible through the engineering of antibodies on the surface of filamentous phage. Antibodies were isolated from a large repertoire of single chain Fv fragments (scFv) derived from non-immunised human donors, with selection and screening procedures biased to favour those binding to free oestradiol. This resulted in an antibody with nanomolar affinity for oestradiol, while affinities for related steroids are in the micromolar range. The relative lack of reactivity for steroids substituted at either end of the molecule suggests that this antibody is unique among anti-steroid monoclonal antibodies in lacking a 'blind-spot'. Our results demonstrate that phage display can provide solutions to problems that have so far proved intractable using conventional hybridoma technology.

Antibodies, Monoclonal↗

PubMed1995

Epitope mapping of anti-HIV and anti-HCV monoclonal antibodies and characterization of epitope mimics using a filamentous phage peptide library.

A large filamentous phage library (1 x 10(9) clones) displaying random 30-amino-acid (aa) sequences on the N terminus of the pIII coat protein was constructed and characterized. Clones in the library were affinity selected for binding to monoclonal antibodies (mAb) against two viral antigens, the HIV gp120 protein and the HCV core protein. The obtained aa sequences precisely identified the epitopes recognized by the mAb. Binding of peptide-carrying phages to the Ab was demonstrated by ELISA, Western blot and the surface plasmon resonance (SPR) method. The mAb-specific peptides were transferred via genetic techniques onto the N terminus of Escherichia coli alkaline phosphatase (AP). When fused to the enzyme, the peptides maintained their ability to bind their respective mAb, indicating that the peptides contained the necessary contact residues for binding. The affinity of the peptides was estimated to be 100 nM by SPR. A comparison is presented of the relative affinities of phage-derived peptides to the native viral epitopes also displayed on the AP scaffold. The approach of transferring epitopes from phage to AP for further evaluation should be applicable to many other mAb or receptors.

Acid Phosphatase↗

PubMed1995

Occurrence, solution structure and stability of DNA hairpins stabilized by a GA/CG helix unit.

The occurrence and NMR solution structure of a class of biloop hairpins containing the sequence 5'-CGXYAG are presented. These hairpins, which are variations on a sequence found in the reverse transcript of the human T-cell leukemia virus 2 (HLV2), show elevated melting points and high chemical stability toward denaturation by urea. Hairpins with the 5'-CGXYAG configuration have melting points 18-20 degrees higher than hairpins with 5'-CAXYGG or 5'-GGXYAC configurations. The identities of the looping bases, X and Y above, play a negligible role in determining the stability of this DNA hairpin stability. This is very different from G-A based loops in RNA, where the third base must be a purine for high stability [the GNRA loops; V.P. Antao, S.Y. Lai and I. Tinoco, Jr (1991) Nucleic Acids Res., 19, 5901-5905]. We show that these properties are associated with a four base helix unit that contains both a sheared GA base pair and a Watson-Crick CG base pair upon which it is stacked. As an understanding of the significance of AG base pairs has become increasingly important in the structural biology of nucleic acids, we compute an 0.7-0.9 A precision ensemble of NMR solution structures using iterative relaxation matrix methods. Calculations performed on NMR-derived structures indicate that neither base-base electrostatic interactions, nor base-solvent dispersive interactions, are significant factors in determining the observed differences in hairpin stability. Thus the stability of the 5'-CGXYAG configuration would appear to derive from favorable base-base London/van der Waals interactions.

Base Composition↗

PubMed1995

Crystallographic analysis of reversible metal binding observed in a mutant (Asp153-->Gly) of Escherichia coli alkaline phosphatase.

Here we present the refined crystal structures of three different conformational states of the Asp153-->Gly mutant (D153G) of alkaline phosphatase (AP), a metalloenzyme from Escherichia coli. The apo state is induced in the crystal over a 3 month period by metal depletion of the holoenzyme crystals. Subsequently, the metals are reintroduced in the crystalline state in a time-dependent reversible manner without physically damaging the crystals. Two structural intermediates of the holo form based on data from a 2 week (intermediate I) and a 2 month soak (intermediate II) of the apo crystals with Mg2+ and Zn2+ have been identified. The three-dimensional crystal structures of the apo (R = 18.1%), intermediate I (R = 19.5%), and intermediate II (R = 19.9%) of the D153G enzyme have been refined and the corresponding structures analyzed and compared. Large conformational changes that extend from the mutant active site to surface loops, located 20 A away, are observed in the apo structure with respect to the holo structure. The structure of intermediate I shows the recovery of the entire enzyme to an almost native-like conformation, with the exception of residues Asp 51 and Asp 369 in the active site and the surface loop (406-410) which remains partially disordered. In the three-dimensional structure of intermediate II, both Asp 51 and Asp 369 are essentially in a native-like conformation, but the main chain of residues 406-408 within the loop is still not fully ordered. The D153G mutant protein exhibits weak, reversible, time dependent metal binding in solution and in the crystalline state.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed1995

A mathematical model for biopanning (affinity selection) using peptide libraries on filamentous phage.

A mathematical model is presented for the process of selection of peptides for binding to a target. The affinity enrichment process commonly known as biopanning relies on subjecting a library of peptides on filamentous phage to a selection for binding to the target immobilized on solid support or in solution. The model is an implementation of the mass-action law to the diverse population of macromolecular assemblies. An analytical solution is presented for the enrichment process. Most parameters in the enrichment formula can be easily determined experimentally. Two examples corresponding to biopanning with epitope libraries and antibody libraries are given. The model allows for an estimation of the contribution of equilibrium and dissociative biopanning to the overall enrichment. The model can be a tool in the evaluation of the role of different biopanning parameters. Its implementation in a spreadsheet makes it possible to perform a computer simulation of a biopanning experiment.

Antibodies↗

PubMed1995

A molecular sensor system based on genetically engineered alkaline phosphatase.

Binding and signaling proteins based on Escherichia coli alkaline phosphatase (AP; EC 3.1.3.1) were designed for the detection of antibodies. Hybrid proteins were constructed by using wild-type AP and point mutants of AP [Asp-101 --> Ser (D101S) and Asp-153 --> Gly (D153G)]. The binding function of the hybrid proteins is provided by a peptide epitope inserted between amino acids 407 and 408 in AP. Binding of anti-epitope antibodies to the hybrid proteins modulates the enzyme activity of the hybrids; upon antibody binding, enzyme activity can increase to as much as 300% of the level of activity in the absence of antibody or can decrease as much as 40%, depending on the presence or absence of the point mutations in AP. The fact that modulation is altered from inhibition to activation by single amino acid changes in the active site of AP suggests that the mechanism for modulation is due to structural alterations upon antibody binding. Modulation is a general phenomenon. The properties of the system are demonstrated by using two epitopes, one from the V3 loop of human immunodeficiency virus type 1 gp120 protein and one from hepatitis C virus core protein, and corresponding monoclonal antibodies. The trend of modulation is consistent for all hybrids; those in wild-type AP are inhibited by antibody, while those in the AP mutants are activated by antibody. This demonstrates that modulation of enzyme activity of the AP-epitope hybrid proteins is not specific to either a particular epitope sequence or a particular antibody-epitope combination.

Alkaline Phosphatase↗

PubMed1995

Expression in Escherichia coli and affinity purification of a CKS-troponin I fusion protein.

The human cardiac troponin I gene was subcloned and expressed at high levels in Escherichia coli as a fusion protein to CMP-KDO synthetase (CKS). Expression levels of the CKS-troponin I fusion were 8% of total cellular protein 4 h after induction with IPTG. The fusion was expressed primarily as an insoluble protein as shown by SDS-PAGE analysis. Expressed CKS-troponin I fusion from a crude lysate was antigenic against anti-CKS and anti-troponin I monoclonal antibodies in Western blots. The fusion was affinity-purified over a TnC affinity column using a urea-solubilized extract of a crude cell lysate. Serial dilutions of crude soluble extracts of the troponin I fusion were assayed in several microparticle enzyme immunoassays and found to exhibit similar immunogenic responses relative to cardiac troponin I isolated from human heart tissue.

Biomarkers↗

PubMed1995

3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity.

The substitution of aspartate at position 153 in Escherichia coli alkaline phosphatase by glycine results in a mutant enzyme with 5-fold higher catalytic activity (kcat) but no change in Km at pH 8.0 in 50 mM Tris-HCl. The increased kcat is achieved by a faster release of the phosphate product as a result of the lower phosphate affinity. The mutation also affects Mg2+ binding, resulting in an enzyme with lower metal affinity. The 3-D X-ray structure of the D153G mutant has been refined at 2.5 A to a crystallographic R-factor of 16.2%. An analysis of this structure has revealed that the decreased phosphate affinity is caused by an apparent increase in flexibility of the guanidinium side chain of Arg166 involved in phosphate binding. The mutation of Asp153 to Gly also affects the position of the water ligands of Mg2+, and the loop Gln152-Thr155 is shifted by 0.3 A away from the active site. The weaker Mg2+ binding of the mutant compared with the wild type is caused by an altered coordination sphere in the proximity of the Mg2+ ion, and also by the loss of an electrostatic interaction (Mg2+.COO-Asp153) in the mutant. Its ligands W454 and W455 and hydroxyl of Thr155, involved in the octahedral coordination of the Mg2+ ion, are further apart in the mutant compared with the wild type.

Alkaline Phosphatase↗

PubMed1994

Modulation of enzyme activity by antibody binding to an alkaline phosphatase-epitope hybrid protein.

An epitope from the HIV-1 gp120 protein V3 loop has been inserted onto the surface of bacterial alkaline phosphatase at different positions in the vicinity of the enzyme active site, creating hybrid proteins that can bind to an anti-gp120 monoclonal antibody. One of the hybrid proteins, API1, has a 13 amino acid V3 loop sequence inserted between residues 407 and 408 of alkaline phosphatase. The enzymatic activity of this protein is modulated upon antibody binding. API1 maintains the full activity of the wild type alkaline phosphatase but in the presence of the anti-gp120 antibody, the enzyme activity is inhibited by 40-50%. Thus, the hybrid enzyme can be used to detect the presence of antibody in solution. The concept of signalling proteins may have a wide application. Two models for the mechanism of modulation, steric hindrance and allosteric regulation, are discussed.

Alkaline Phosphatase↗

PubMed1992

3-D structure of a mutant (Asp101-->Ser) of E.coli alkaline phosphatase with higher catalytic activity.

Mutagenesis of the absolutely conserved residue Asp101 of the non-specific monoesterase alkaline phosphatase (E.C. 3.1.3.1) from E. coli has produced an enzyme with increased kcat. The carboxyl group of the Asp101 residue has been proposed to be involved in the positioning of Arg166 and the formation of the helix that contains the active site Ser102. The crystal structure of the Asp101-->Ser mutant has been refined at 2.5 A to a final crystallographic R-factor of 0.173. The altered active site structure of the mutant is compared with that of the wild-type as well as with the structures of the mutant enzyme soaked in two known alkaline phosphatase inhibitors (inorganic phosphate and arsenate). The changes affect primarily the side chain of Arg166 which, by losing the hydrogen bond interaction with the carboxyl side chain of Asp101, becomes more flexible. This analysis, in conjunction with product inhibition studies of the mutant enzyme, suggests that at high pH (> 7) the enzyme achieves a quicker catalytic turnover by allowing a faster release of the product.

Alkaline Phosphatase↗

PubMed1991

Preparation of synthetic polypeptide domains of carcinoembryonic antigen and their use in epitope mapping.

Genes encoding the four principal polypeptide domains (N, A1-B1, A2-B2, and A3-B3) of carcinoembryonic antigen (CEA) were synthesized and expressed in Escherichia coli as fusion products with bacterial CMP-KDO synthetase (CKS). The four synthetic fusion proteins were purified in high yield and used as targets in Western blots for 11 anti-CEA MAbs and to compete with immobilized CEA for binding to four of these MAbs. Each of the MAbs showed strong binding to one or more of the fusion proteins. In Western blots, MAbs H19C91 and 4230 bound only to CKS-N. MAbs H8C2 and H11C35 bound only CKS-A1-B1, and MAbs T84.66, H46C136, and H21C83 appeared to be specific for CKS-A3-B3. None of the MAbs tested bound only to CKS-A2-B2. However, two MAbs bound both CKS-A1-B1 and CKS-A3-B3 and one MAb (3519) bound to all three of the repeated domains. Since these three domains exhibit over 90% amino acid sequence homology, the latter results were not surprising. The competition studies largely confirmed the results of Western blots but did show some MAb-fusion protein interactions not observed in Western blots. These competition studies also allowed estimation of the relative affinities of the MAbs for the synthetic domains and for native CEA. These studies demonstrated that epitopes in CEA recognized by the MAbs in this study are peptide in nature and that the fusion proteins are of utility in the localization of the epitopes on the polypeptide chain of CEA.

Antibodies, Monoclonal↗

PubMed1991

Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat.

The likelihood for improvement in the catalytic properties of Escherichia coli alkaline phosphatase was examined using site-directed mutagenesis. Mutants were constructed by introducing sequence changes into nine preselected amino acid sites within 10 A of the catalytic residue serine 102. When highly conserved residues in the family of alkaline phosphatases were mutated, many of the resulting enzymes not only maintained activity, but also exhibited greatly improved kcat. Of approximately 170 mutant enzymes screened, 5% (eight mutants) exhibited significant increases in specific activity. In particular, a substitution by serine of a totally invariant Asp101 resulted in a 35-fold increase of specific activity over wild-type at pH 10.0. Up to 6-fold increases of the kcat/Km ratio were observed.

Alkaline Phosphatase↗

PubMed1991

C5a structural requirements for neutrophil receptor interaction.

A number of C5a modifications were tested to determine effects on receptor binding to polymorphonuclear leukocyte (PMNL) membrane receptors and triggering of PMNL chemokinesis and myeloperoxidase (MPO) release. Site-directed mutagenesis was used to probe relationships of key C-terminal residues, and suggested a role for additional sites, particularly Lys19-20. A synthetic peptide based on C5a 19-30, weakly inhibited C5a binding. Potency of the C-terminal octapeptide, a full agonist, was markedly improved by a single Phe substitution for His67, and a Phe point mutation at this site was shown to enhance activity of the full recombinant protein.

Cells, Cultured↗

PubMed1990

A totally synthetic plasmid for general cloning, gene expression and mutagenesis in Escherichia coli.

A first totally synthetic Escherichia coli plasmid has been designed, constructed and shown to be a functional, stable, high-copy cloning vector. The FokI method of gene synthesis [Mandecki and Bolling, Gene 68 (1988) 101-107] was used to assemble the plasmid from 30 oligodeoxyribonucleotides. The plasmid contains synthetic modules for the beta-lactamase-encoding gene (bla), replication origin, lacZ gene fragment and multicloning site. The plasmid is patterned after the pUC-type plasmids and has a copy number similar to that of pUC plasmids. The major changes introduced include the removal of nearly 50% of the restriction sites present in pUC plasmids, reduction of plasmid size to 2050 bp, and introduction of transcription terminators downstream from both the bla gene and lacZ fragment. The changes facilitate a number of techniques, such as cloning, mutagenesis, expression and restriction analysis.

Base Sequence↗