Glenn G. Lilley

The three-dimensional structure of N -acetylneuraminate lyase from Escherichia coli

BACKGROUND N-acetylneuraminate lyase catalyzes the cleavage of N-acetylneuraminic acid (sialic acid) to form pyruvate and N-acetyl-D-mannosamine. The enzyme plays an important role in the regulation of sialic acid metabolism in bacteria. The reverse reaction can be exploited for the synthesis of sialic acid and some of its derivatives. RESULTS The structure of the enzyme from Escherichia coli has been determined to 2.2 A resolution by X-ray crystallography. The enzyme is shown to be a tetramer, in which each subunit consists of an alpha/beta-barrel domain followed by a carboxy-terminal extension of three alpha-helices. CONCLUSIONS The active site of the enzyme is tentatively identified as a pocket at the carboxy-terminal end of the eight-stranded beta-barrel. Lys165 lies within this pocket and is probably the reactive residue which forms a Schiff base intermediate with the substrate. The sequence of N-acetylneuraminate lyase has similarities to those of dihydrodipicolinate synthase and MosA (an enzyme implicated in rhizopine synthesis) suggesting that these last two enzymes share a similar structure to N-acetylneuraminate lyase.

Use of mutator cells as a means for increasing production levels of a recombinant antibody directed against Hepatitis B

A mutation strategy which utilises phage display technology and the Escherichia coli mutator strains, mutD5-FIT and XL1-RED, was applied to a Hepatitis B (HepB) specific single-chain Fv (scFv) to incorporate random mutations throughout the gene. Messenger RNA from a hybridoma producing antibodies against HepB was isolated, reverse transcribed and used as template for the production of scFv. Following production of the scFv protein using an E. coli expression vector (pGC), the scFv gene was recloned into a phage display vector (pHFA). This gene construct was introduced into E. coli mutator cells and the transformed cells were used as an inoculum for liquid cultures. After five cycles of growth at 37 degrees C, each followed by dilution and re-inoculation of fresh media, recombinant phage were recovered. Nucleotide sequence analysis of the scFv gene in phage selected on HBsAg-coated magnetic beads identified amino acid substitutions which produced an increase of greater than 10-fold in apparent production levels. Competitive ELISA studies showed that the selected scFv mutants appeared to have similar affinity to HBsAg as the parent scFv. The apparent increase in production was not the result of improved surface characteristics of regions uniquely exposed in scFvs, as the sites did not correlate with the variable/constant interface of the scFv variable region normally masked in Fabs or IgGs.

Recombinant single-chain antibody peptide conjugates expressed in Escherichia coli for the rapid diagnosis of HIV

Recombinant single chain Fv (scFv) antibody fragments can form the basis of a rapid, whole-blood diagnostic assay. The scFv described in this study is derived from a monoclonal antibody which has a high affinity for glycophorin A, an abundant glycoprotein on the human red blood cell membrane surface. The prototype reagent built around the scFv was designed to detect, in whole blood samples, the presence of antibodies that have arisen through infection with a foreign organism such as human immunodeficiency virus. The scFv was composed of the antibody heavy-chain variable domain (Vh) joined by a 15 residue linker -(GGGGS)3- to the light-chain variable domain (V1) terminated by either a C-terminal octapeptide tail (FLAG) or a 35 amino acid segment from the gp41 surface glycoprotein of HIV-1. Constructs were cloned into a Escherichia coli expression vector, pHFA, and expressed in a soluble form into culture supernatant. The product retained anti-glycophorin activity which could be detected directly in culture supernatants by ELISA. Furthermore, the scFv-epitope fusion functioned efficiently in the whole blood agglutination assay and was able to distinguish between HIV-1 positive and negative sera.

Construction of recombinant extended single-chain antibody peptide conjugates for use in the diagnosis of HIV-1 and HIV-2

The construction, expression and evaluation of recombinant scFv based HIV diagnostic reagents are described. In a whole-blood, erythrocyte agglutination assay format, recombinant scFv antibodies (expressed in Escherichia coli), linked to a spacer domain and HIV-gp36 or -gp41 peptides, were shown to be able to detect efficiently natural antibodies against HIV in human serum. Performance in trials suggests that these single chain reagents have potential as alternatives to existing Fab-peptide chemical conjugates. We also report the construction of an inducible expression vector, pGC, which can be used both in laboratory experiments and in large-scale fed-batch fermentations. It was found that while the base scFv reagent (lacking a spacer) functioned as well as the Fab peptide conjugate in assays where whole (negative) blood was spiked with mouse monoclonal anti-HIV antibodies (IgG or IgM), clinical assays using human sera showed lower sensitivities and increased false negatives. This deficiency was overcome by inclusion of the natural 1C3 kappa (light) chain domain as a spacer arm between the scFv and HIV peptide tags. This spacer was thought to overcome steric constraints which would otherwise prevent efficient interaction between the reagent (once bound to the surface of red blood cells) and the various serum antibodies against the respective C-terminal peptide epitopes. As a result of this important modification, performance of the extended scFv reagent (for both HIV-1 and HIV-2) equalled that of the current commercial technology in limited trials.

Amino acid sequence of conglutin δ, a sulfur-rich seed protein of Lupinus angustifolius L.: Sequence homology with the C-III α-amylase inhibitor from wheat

Complete amino acid sequences and disulfide cross‐link arrangements have been determined for the two subunit polypeptides (M r 9401 and 4597) ofconglutin δ, a helix‐rich seed protein from Lupinus angustifolius cv. Uniwhite. There are two intrachain disulfide bonds and a free sulfhydryl group within the large chain and two interchain disulfide bonds to the small chain. The sequences show regions enriched in glutamineglutamic acid and serine residues which were correlated by a predictive method to the high measured level of α‐helix (~ 38%). Homology was found between a cystine‐rich region ofconglutin δ and the C‐III α‐amylase inhibitor from wheat suggesting that these proteins originated from a common ancestral gene.

Antigen binding and cytotoxic properties of a recombinant immunotoxin incorporating the lytic peptide, melittin

BACKGROUND The majority of immunotoxins studied to date incorporate toxins that act in the cytosol and thus need to be endocytosed by the target cell. An alternative strategy for immunotoxin development is the use of membrane active toxins, such as the pore-forming proteins. Melittin, a 26 amino acid cytolytic peptide from bee venom, is such a protein. OBJECTIVES We report here the construction, production and functional analysis of a recombinant immunotoxin obtained by fusion of genes which encode an antibody fragment (scFv) with an oligonucleotide encoding melittin. STUDY DESIGN The antibody fragment was derived from a murine monoclonal antibody, K121, which recognises a specific epitope (KMA) expressed on the surface of human kappa myeloma and lymphoma cells, and on human free kappa Bence Jones protein (BJP). Melittin is a 26-amino acid, membrane-lytic peptide which is a major component of bee venom. The scFv of K121 was constructed by PCR to link VH and VL genes via an oligonucleotide which encodes a flexible, hydrophilic peptide. An oligonucleotide encoding melittin and the peptide marker sequence FLAG was fused to the scFv construct using a similar linker peptide. The gene construct (scFv-mel) was inserted into the secretion vector pPOW and expressed in Escherichia coli (TOPP2). RESULTS Expression of the recombinant scFv-mel gene and purification of the protein product was monitored by Western blot analysis. Following purification by anti-FLAG affinity chromatography, the recombinant immunotoxin (scFv-mel) was assessed for antigen binding and for cytotoxic activity by flow cytometry using antigen-expressing and non-expressing cell targets. The scFv-mel was found to exhibit binding and killing properties consistent with the specificity of the original K121 antibody. Moreover, the cytolytic activity of the scFv-mel was significantly greater on a molar basis than that of native melittin alone. CONCLUSION The data presented here constitute the first report of a melittin-based recombinant immunotoxin and demonstrate that such a membrane active immunotoxin can be synthesised in a bacterial expression. Linking of melittin to an antibody fragment overcame the non-specific toxicity of melittin as the recombinant immunotoxin exhibited specific toxicity towards antigen-bearing target cells. The observation that the immunotoxin exhibited enhanced cytotoxic activity over the free toxin indicates the potential of this approach for the development of an effective therapeutic agent.

Biosynthesis, cDNA and amino acid sequences of a precursor of conglutin δ, a sulphur-rich protein from Lupinus angustifolius.

The biosynthesis of conglutin δ has been studied in developing cotyledons of Lupinus angustifolius L. Precursors of conglutin δ formed the major sink for [35S]-cysteine incorporated by developing lupin cotyledons, and these precursors were rapidly sequestered into the endoplasmic reticulum. The sequence of a cDNA clone coding for one such precursor of conglutin δ was determined. The structure of the precursor polypeptide for conglutin δ predicted from the cDNA sequence contained an N-terminal leader peptide of 22 amino acids directly preceding a subunit polypeptide of Mr 4520, together with a linking region of 13 amino acids and a subunit polypeptide of Mr 9558 at the C-terminus. The amino acid sequence predicted from the cDNA sequence showed minor variations from that established by sequencing of the protein purified from mature dried seeds (Lilley and Inglis, 1986). These were consistent with the existence of a multi-gene family coding for conglutin δ. Comparison of the sequences of conglutin δ with those of other 2S storage proteins showed that the cysteines involved in internal disulphide bridges between the mature subunits of conglutin δ, were maintained throughout this family of proteins but that little else was conserved either at the protein or DNA level.

Crystalline monoclonal Fab fragment with specificity towards an influenza virus neuraminidase

Abstract An Fab fragment from a monoclonal antibody (NTS10/1) has been crystallized. The antigen, influenza virus A/Tokyo/3/67 (N2) neuraminidase, is also crystalline and its structure analysis is in progress. The Fab crystals are trigonal, space group P3121 with cell dimensions a = 132·3 A , c = 73·8 A . This crystalline material forms a complex with the neuraminidase with an equilibrium binding constant in excess of 1011 m −1. The molecular weight of the complex is 406,000 ± 20,000 indicating that four Fab fragments are attached to each neuraminidase tetramer. The separate crystallization of antigen and Fab fragment opens the way to map, for the first time, the complementary surfaces of an antigen-antibody complex.

Structural studies of a french bean storage protein: phaseolin

Molecular weights and sedimentation coefficients have been measured for different oligomeric forms of phaseolin, the major storage protein in seeds of Phaseolus vulgaris L. The results indicate that phaseolin is a trimer (Mr = 150000) at neutral pH which aggregates further to a dodecamer form (Mr = 596000) at pH 4.5. The subunit size is in good agreement with the recently determined sequence molecular weight, if allowance is made for bound oligosaccharide and phytic acid moieties. The trimeric nature at neutral pH has been confirmed by chemical crosslinking studies using dimethylsuberimidate and dithiobis(succinimidylpropionate). Analyses of optical rotatory dispersion and circular dichroism data have been used to examine the corformation of phaseolin. In common with other seed globulins, a low proportion of α-helix (∼ 10%) coupled with a high level of β-sheet (∼50%) is predicted. These data are compared with a structural analysis based on the amino acid sequence of a phaseolin subunit polypeptide. The predicted level of α-helix is increased (∼20%) when phaseolin is heated in sodium dodecyl sulphate, but not when the detergent is added at room temperature.

Escherichia coli expression of a bifunctional Fab-peptide epitope reagent for the rapid diagnosis of HIV-1 and HIV-2

BACKGROUND The current format of a rapid whole-blood agglutination assay for HIV relies on a bifunctional molecule which comprises a 1C3 Fab fragment, with specificity for the human red blood cell surface marker (glycophorin A), chemically conjugated to a synthetic peptide that corresponds to a single immunodominant region of HIV envelope glycoprotein. In this assay erythrocyte agglutination occurs if the blood sample contains anti-HIV antibodies. OBJECTIVES To establish whether a bacterially synthesised Fab fragment encoding several C-terminal immunodominant peptide tails can be produced in sufficient purity and yield to function in whole-blood agglutination assays. STUDY DESIGN An E. coli dicistronic Fab expression cassette was constructed comprising of light and heavy chain gene fragments derived from a glycophorin specific monoclonal antibody (1C3), genetically linked with C-terminal immunoreactive peptide epitopes. Expression and purification procedures were established to enable the rapid production of 1C3 Fab-peptide epitope conjugates. RESULTS A recombinant 1C3 Fab fragment was expressed with two different immunological epitope markers, Glu-Glu-Phe (EEF) and FLAG, at the C-terminus of the Fd heavy and kappa light chain, respectively. This model Fab-EEF/FLAG conjugate was detected in culture supernatant by SDS-PAGE gels and Western blots, and could be successfully used in erythrocyte agglutination assays. Furthermore, an HIV specific 1C3 Fab reagent, containing immunoreactive peptide epitopes from the surface glycoproteins of HIV-1 and HIV-2, was also expressed but at lower levels and with increased sensitivity to proteolytic degradation. Nevertheless, this recombinant Fab reagent with dual diagnostic specificity performed very effectively in whole-blood diagnosis of patients infected with either HIV-1 or HIV-2. CONCLUSION A recombinant 1C3 Fab fragment terminated by immunoreactive peptide epitopes can be expressed in E. coli in a soluble, antigen-binding form, and it can successfully mimic the commercial Fab-HIV reagents in whole-blood agglutination assays.