Structural analysis of the carboxy terminus of bacteriophage lambda repressor determined by antipeptide antibodies.

To analyze lambda repressor function and structure, antibodies were generated with synthetic peptides corresponding to sequences believed to be involved in prophage induction. These site-directed antibodies seemed to recognize preferentially the primary sequence of repressor because they reacted better in competition experiments with the oligopeptide and with the partially denatured forms of repressor than with the native molecules. This information, together with the characteristic ability of the antibodies to immunoprecipitate or react with repressor in immunoblots, allowed us to infer some conformational properties of the specific regions that the antibodies recognized. The antibodies reacted less with some mutant repressors that had a single amino acid substitution within the cognitive sequences. RecA-catalyzed cleavage of repressor was inhibited to different extents in relation to the proportion of repressor that each antipeptide immunoglobulin G (IgG) was able to immunoprecipitate. The antipeptide IgGs did not affect specific binding of repressor to operator DNA, whereas the antirepressor IgG was inhibitory. The three different IgGs competed for binding to repressor in an enzyme-linked immunosorbent assay additivity test, which suggested that the three regions of conserved amino acids are probably located on the same side of the carboxyl domain of repressor and possibly close together in the tertiary structure.     

High-density functional display of proteins on bacteriophage lambda

We designed a bacteriophage lambda system to display peptides and proteins fused at the C terminus of the head protein gpD of phage lambda. DNA encoding the foreign peptide/protein was first inserted at the 3' end of a DNA segment encoding gpD under the control of the lac promoter in a plasmid vector (donor plasmid), which also carried lox P(wt) and lox P(511) recombination sequences. Cre-expressing cells were transformed with this plasmid and subsequently infected with a recipient lambda phage that carried a stuffer DNA segment flanked by lox P(wt) and lox P(511) sites. Recombination occurred in vivo at the lox sites and Amp(r) cointegrates were formed. The cointegrates produced recombinant phage that displayed foreign protein fused at the C terminus of gpD. The system was optimised by cloning DNA encoding different length fragments of HIV-1 p24 (amino acid residues 1-72, 1-156 and 1-231) and the display was compared with that obtained with M13 phage. The display on lambda phage was at least 100-fold higher than on M13 phage for all the fragments with no degradation of displayed products. The high-density display on lambda phage was superior to that on M13 phage and resulted in selective enrichment of epitope-bearing clones from gene-fragment libraries. Single-chain antibodies were displayed in functional form on phage lambda, strongly suggesting that correct disulphide bond formation takes place during display.This lambda phage display system, which avoids direct cloning into lambda DNA and in vitro packaging, achieved cloning efficiencies comparable to those obtained with any plasmid system. The high-density display of foreign proteins on bacteriophage lambda should be extremely useful in studying low-affinity protein-protein interactions more efficiently compared to the M13 phage-based system.     

Outer surface protein of bacteriophage lambda

The bacteriophage λ capsid is composed of a main shell protein (pE) and an outer surface protein (pD). The outer surface protein was purified from sources of free protein and assembled protein. The amino acid composition, C- and N-terminals, iso-electric point, molecular weight, and state of aggregation were determined. In vitro the outer surface protein binds specifically to structures composed of λ main shell protein in the expanded configuration i.e. to enlarged preheads, pD-deficient bacteriophage particles, and polyheads.We discuss the binding of pD to the shell surface as a “pseudo-crystallisation process”, its clustering on the surface as trimers and its role as stabiliser of the filled head.     

Structural basis of bacteriophage lambda capsid maturation

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Isolating and engineering human antibodies using yeast surface display

This protocol describes the process of isolating and engineering antibodies or proteins for increased affinity and stability using yeast surface display. Single-chain antibody fragments (scFvs) are first isolated from an existing nonimmune human library displayed on the yeast surface using magnetic-activated cell sorting selection followed by selection using flow cytometry. This enriched population is then mutagenized, and successive rounds of random mutagenesis and flow cytometry selection are done to attain desired scFv properties through directed evolution. Labeling strategies for weakly binding scFvs are also described, as well as procedures for characterizing and 'titrating' scFv clones displayed on yeast. The ultimate result of following this protocol is a panel of scFvs with increased stability and affinity for an antigen of interest.     

Topography of bacteriophage T7 RNA polymerase using monoclonal antibodies]

Four clones producing monoclonal antibodies inhibiting enzymatic activity were used to localize the functionally important antigenic determinants of T7 RNA polymerase. All antibodies were shown to bind to C-terminal fragment of the protein (residues 589-883). The competition studies showed the specificity of the three antibodies toward one epitope and the fourth antibody to another one. By means of limited cleavage of the RNA polymerase with cyanogen bromide with subsequent electrophoretic separation and immunoblotting the peptides containing antigenic determinants were localized. These are Met861-Ala883 for antibody 4H8 and Met750-Met832 for antibodies 9B2, 3H11 and 2A2.     

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Phage peptide libraries constitute powerful tools for themapping of epitopes recognized by monoclonal antibodies (mAbs).Using screening of phage displayed random peptide libraries wehave characterized the binding epitopes of three mAbs directedagainst the surface envelope glycoprotein (gp46) of the humanT-cell leukemia virus type I (HTLV-I). Two phage libraries,displaying random heptapeptides with or without flankingcysteine residues, were screened for binding to mAbs 7G5D8, DB4and 4F5F6. The SSSSTPL consensus sequence isolated fromconstrained heptapeptide library defines the epitope recognizedby DB4 mAb and corresponds to the exact region 249–252 of thevirus sequence. The APPMLPH consensus sequence isolated fromnon constrained heptapeptide library defines the epitoperecognized by 7G5D8 mAb and corresponds to the region 187–193with a single amino acid substitution, methionine to leucine atposition 190. The third consensus sequence LYWPHD isolated fromconstrained heptapeptide library defines the epitope recognizedby 4F5F6 mAb. It corresponds to an epitope without directequivalence with the virus sequence. The data presented hereshowed that 7G5D8 and DB4 mAbs are raised against linearepitopes while 4F5F6 mAb recognized a continuous topographic epitope.     

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Summary Phage peptide libraries constitute powerful tools for the mapping of epitopes recognized by monoclonal antibodies (mAbs). Using screening of phage displayed random peptide libraries we have characterized the binding epitopes of three mAbs directed against the surface envelope glycoprotein (gp46) of the human T-cell leukemia virus type I (HTLV-I). Two phage libraries, displaying random heptapeptides with or without flanking cysteine residues, were screened for binding to mAbs 7G5D8, DB4 and 4F5F6. The SSSSTPL consensus sequence isolated from constrained heptapeptide library defines the epitope recognized by DB4 mAb and corresponds to the exact region 249–252 of the virus sequence. The APPMLPH consensus sequence isolated from non constrained heptapeptide library defines the epitope recognized by 7G5D8 mAb and corresponds to the region 187–193 with a single amino acid substitution, methionine to leucine at position 190. The third consensus sequence LYWPHD isolated from constrained heptapeptide library defines the epitope recognized by 4F5F6 mAb. It corresponds to an epitope without direct equivalence with the virus sequence. The data presented here showed that 7G5D8 and DB4 mAbs are raised against linear epitopes while 4F5F6 mAb recognized a continoous topographic epitope.     

Succinic acid production from sucrose and molasses by metabolically engineered E. coli using a cell surface display system

To achieve sucrose-metabolizing capability, different sucrose utilization operons have been introduced into E. coli that cannot utilize sucrose. However, these engineered strains still suffer from low growth rates and low sucrose uptake rates. In this study, cell surface display system was adopted in engineered E. coli AFP111 for succinic acid production from sucrose and molasses directly. Invertase (CscA) from E. coli W was successfully anchored to outer membrane by fusion with OmpC anchoring motif, and the displayed CscA showed high extracellular activity. Compared with the sucrose permease system, the cell surface display system consumed less ATP during sucrose metabolism. When less ATP was consumed by AFP111/pTrcC-cscA, the succinic acid productivity from sucrose was 23% higher than that by AFP111/pCR2.1-cscBKA that having the sucrose permease system. As a result, 41 g L⁻¹ and 36.3 g L⁻¹ succinic acid were produced by AFP111/pTrcC-cscA from sucrose and sugarcane molasses respectively at 34 h in 3-L fermentor during dual-phase fermentation. In addition, 79 g L⁻¹ succinic acid was accumulated with recovered AFP111/pTrcC-cscA cells at the end of dual-phase fermentation in 3-L fermentor, and the overall yield was 1.19 mol mol⁻¹ hexose.     

Analysis of murine coronavirus surface glycoprotein functions by using monoclonal antibodies.

The murine coronavirus surface glycoprotein gene was expressed as a fusion protein in bacteria, and the expressed protein was used to generate S protein-specific monoclonal antibodies (MAbs). Three of the MAbs, 11F, 30B, and 10G, were able to neutralize virus infectivity, and two of them, 11F and 10G, were able to block virus-induced, cell-to-cell fusion. The binding sites of the 11F, 30B, and 10G MAbs were determined by Western immunoblotting and epitope mapping. The 11F and 30B MAbs bound to sites located, respectively, between amino acids 33 to 40 and 395 to 406 in the amino-terminal (S1) subunit of the S protein, and the 10G MAb bound to a site located between amino acids 1123 and 1137 in the carboxy-terminal (S2) subunit. These data define more precisely the interactions between the S1 and S2 subunits of the murine coronavirus S protein and provide further insights into its structure and function.     

Structural studies of bacteriophage lambda heads and proheads by small angle X-ray diffraction.

We have examined a series of lambda proheads and mature structures by small angle X-ray diffraction. This technique yields spherically averaged density distributions and some information about surface organization of particles in solution.We find that gpE ² of proheads and heads forms shells with one of two radii; A^?, B^?, groE^?, and Nu3^? proheads have shells of radius 246 Å, while mature heads, urea-treated A^? proheads and C^? proheads have a radius of 300 Å. The expansion of proheads to mature heads is accompanied by a corresponding decrease in the thickness of the shell. groE^? proheads contain a core. This core is lost spontaneously from the structure and is only observed if the structures are fixed with glutaraldehyde prior to examination by X-ray diffraction or electron microscopy.C^? proheads expand to mature head size spontaneously. A preparation of C^? proheads which was fixed with glutaraldehyde at an early stage of the purification had the smaller, prohead radius. Unfixed particles from this preparation expanded to the mature head size after further purification and standing in the cold for several days. This result suggests that gpC may be involved in regulating head expansion.The radii of the protein shells of mature heads are identical for a series of phages that contain between 78% and 105% of the wild-type complement of DNA, and this radius is the same as that of proheads expanded in the absence of DNA. These results with phage lambda indicate that assembly of a double shell structure composed of coat and scaffolding protein, followed by expansion to a larger shell containing only coat protein is a general feature of the morphogenesis of dsDNA phages.     

Transduction of bacteriophage lambda by bacteriophage T1.

When bacteriophage T1 was grown on bacteriophage lambda-lysogenic cells, phenotypically mixed particles were formed which had the serum sensitivity, host range, and density of T1 but which gave rise to lambda phage. T1 packaged lambda genomes more efficiently both when the length of the prophage was less than that of wild-type lambda and when the host cell was polylysogenic. Expression of the red genes of lambda or the recE system of Escherichia coli during T1 growth enhanced pickup of lambda by T1, whereas packaging was reduced in recB cells. If donors were singly lysogenic, the expression of transduced lambda genomes as a PFU required lambda-specified excisive recombination, whereas lambda genomes transduced from polylysogens required only lambda- or E. coli-specified general recombination to give a productive infection.     

Brewing spoilage Lactobacilli detected using monoclonal antibodies to bacterial surface antigens.

A panel of thirteen monoclonal antibodies (Mabs) was assembled that reacts with surface antigens on eight of eleven Lactobacillus brewing spoilage organisms, including one or more of L. brevis, L. buchneri, L. casei-alactosus, L. plantarum, or unspeciated isolate(s). Immunoblotting was done to identify the antigens involved in Mab binding. Antigen stability in situ was tested by protease treatment and by surface antigen extraction of washed bacteria. Protease susceptibility of extracted surface antigens was also examined. In most cases, Lactobacillus surface antigens detected by the Mabs appear to be noncovalently bound proteins readily altered or removed from the bacterium by various environmental conditions. This research identifies brewing conditions that need to be tested to ascertain whether bacterial surface antigen-reactive Mabs can be used for the rapid, sensitive, and specific detection of Lactobacillus brewing spoilage organisms.     

Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display

Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat. Thus, understanding mechanisms of antibody-mediated viral inhibition and neutralization escape is critical. Here, a robust yeast display system for fine epitope mapping of viral surface hemagglutinin (HA)-specific antibodies is demonstrated. The full-length H5 subtype HA (HA0) was expressed on the yeast surface in a correctly folded conformation, determined by binding of a panel of extensively characterized neutralizing human monoclonal antibodies (mAbs). These mAbs target conformationally-dependent epitopes of influenza A HA, which are highly conserved across H5 clades and group 1 serotypes. By separately displaying HA1 and HA2 subunits on yeast, domain mapping of two anti-H5 mAbs, NR2728 and H5-2A, localized their epitopes to HA1. These anti-H5 mAb epitopes were further fine mapped by using a library of yeast-displayed HA1 mutants and selecting for loss of binding without prior knowledge of potential contact residues. By overlaying key mutant residues that impacted binding onto a crystal structure of HA, the NR2728 mAb was found to interact with a fully surface-exposed contiguous patch of residues at the receptor binding site (RBS), giving insight into the mechanism underlying its potent inhibition of virus binding. The non-neutralizing H5-2A mAb was similarly mapped to a highly conserved H5 strain-specific but poorly accessible location on a loop at the trimer HA interface. These data further augment our toolchest for studying HA antigenicity, epitope diversity and accessibility in response to natural and experimental influenza infection and vaccines.     

Sulfur dioxide inhibits the sucrose carrier of the plant plasma membrane.

Plasma membrane vesicles were prepared by phase partition from a microsomal fraction of broad bean (Vicia faba L.) leaf. In order to study the effects of sodium sulfite on active uptake of sucrose, the vesicles were artificially energized by a transmembrane pH gradient (delta pH) and/or a transmembrane electrical gradient (delta psi). At 1 mM, sulfite strongly inhibited sucrose uptake but did not affect the two components of the proton motive force, delta pH (measured by dimethyloxazolidine dione) and delta psi (measured by tetraphenylphosphonium). Moreover, sulfite did not inhibit the proton-pumping ATPase of the plasma membrane vesicles. These data demonstrate that sulfite may inhibit transport of photoassimilates in plant by a direct inhibition of the sucrose carrier of the plasma membrane.     

Genetic analysis of second-site revertants of bacteriophage lambda integrase mutants.

Bacteriophage lambda site-specific recombination is catalyzed by the phage-encoded integrase (Int) protein. Using a collection of 21 recombination-defective Int mutants, we performed a second-site reversion analysis. One of the primary mutants contained a valine-to-glutamic acid change at position 175 (V175E), and a pseudorevertant with a lysine change at this site (V175K) was also isolated. Relative to the wild-type protein, the V175E protein was defective in its ability to form the attL complex and to catalyze excision in vivo and in vitro. A mutant containing an alanine substitution (V175A) was made by site-directed mutagenesis, and it was more efficient than the V175K protein in forming the attL complex and promoting excision. These results indicate that a nonpolar side chain at residue 175 is required for function. The second primary mutant contained a proline-to-leucine change at position 243 (P243L). A true second-site revertant was isolated that contained a glutamic acid-to-lysine change (E218K). The P243L-E218K protein promoted recombination and bound arm-type sites more efficiently than the original P243L protein but not as efficiently as the protein containing the E218K substitution alone. The E218K substitution also restored activity to a mutant with a threonine-to-isoleucine substitution at position 270 (T270I). This result showed that suppression by the E218K change is not allele specific and suggests that the substitution improves an inherent activity of Int rather than directly compensating for the defect caused by the primary substitutions. Results with challenge phages carrying attL sites with altered core sites indicate that the E218K change may improve binding to the core site.     

Serological analysis of serogroup icterohaemorrhagiae using monoclonal antibodies

Eighteen serovars (19 strains) of serogroup Icterohaemorrhagiae were serologically analyzed using 18 monoclonal antibodies against serovar copenhageni Shiromizu, M20 and serovar icterohaemorrhagiae RGA strains. The reaction patterns of the serovars against these monoclonal antibodies were different. According to these results, we divided the serovars, except for serovar tonkini, into the following three subgroups: Subgroup 1 reacted to many monoclonal antibodies including serovars icterohaemorrhagiae, copenhageni, hualien, monymusk, mankarso, and budapest. Subgroup 2 fell between subgroups 1 and 3 including serovars dakota, naam, bogvere, birkini, smithi, ndambari, gem, ndahambukuje and mwogolo. Subgroup 3 reacted to only a few monoclonal antibodies: serovars weaveri and sarmin. Serovar tonkini did not react to any of the monoclonal antibodies used. There is a possibility that serovar tonkini does not belong to serogroup Icterohaemorrhagiae. Further studies on the serological reactions of each strain revealed that it was impossible to distinguish the RGA strain from the serovar hualien LT11-31 strain, indicating that they may be identical. It was also observed that serovar copenhageni and monymusk seemed to be closely related. Serovars birkini and smithi, and serovars ndambari and gem were alike in their serological reactivities. Among the 18 monoclonal antibodies, RGAMA-1 was a unique antibody which reacted only to serovar icterohaemorrhagiae and serovar hualien, indicating that it must be the serovar icterohaemorrhagiae specific antibody. On the other hand, SHIRMA-2, 5, 6 reacted to all the serovars except for serovars weaveri, sarmin, and tonkini. These antibodies exhibited a broad reaction spectrum.     

Functional analysis of TRAIL receptors using monoclonal antibodies

mAbs were generated against the extracellular domain of the four known TNF-related apoptosis-inducing ligand (TRAIL) receptors and tested on a panel of human melanoma cell lines. The specificity of the mAb permitted a precise evaluation of the TRAIL receptors that induce apoptosis (TRAIL-R1 and -R2) compared with the TRAIL receptors that potentially regulate TRAIL-mediated apoptosis (TRAIL-R3 and -R4). Immobilized anti-TRAIL-R1 or -R2 mAbs were cytotoxic to TRAIL-sensitive tumor cells, whereas tumor cells resistant to recombinant TRAIL were also resistant to these mAbs and only became sensitive when cultured with actinomycin D. The anti-TRAIL-R1 and -R2 mAb-induced death was characterized by the activation of intracellular caspases, which could be blocked by carbobenzyloxy-Val-Ala-Asp (OMe) fluoromethyl ketone (zVAD-fmk) and carbobenzyloxy-Ile-Glu(OMe)-Thr-Asp (OMe) fluoromethyl ketone (zIETD-fmk). When used in solution, one of the anti-TRAIL-R2 mAbs was capable of blocking leucine zipper-human TRAIL binding to TRAIL-R2-expressing cells and prevented TRAIL-induced death of these cells, whereas two of the anti-TRAIL-R1 mAbs could inhibit leucine zipper-human TRAIL binding to TRAIL-R1:Fc. Furthermore, use of the blocking anti-TRAIL-R2 mAb allowed us to demonstrate that the signals transduced through either TRAIL-R1 or TRAIL-R2 were necessary and sufficient to mediate cell death. In contrast, the expression of TRAIL-R3 or TRAIL-R4 did not appear to be a significant factor in determining the resistance or sensitivity of these tumor target cells to the effects of TRAIL.