Mark Suter

Display of biologically active proteins on the surface of filamentous phages: a cDNA cloning system for selection of functional gene products linked to the genetic information responsible for their production.

A cloning and expression system allowing display of functional cDNAs or other gene products on the surface of filamentous phage has been developed, exploiting the high-affinity interaction of the Jun and Fos leucine zippers. Gene jun was expressed from a lacZ promoter as a fusion protein with the viral coat protein, pIII, thereby being structurally incorporated into phage particles during infection with a helper phage. Using a second lacZ promoter of the phagemid, gene fos was co-expressed as an N-terminal fusion peptide to cDNA library gene products, so that the resulting Fos-fusion proteins could become associated with the Jun-decorated phage particles. To avoid interphage exchange of fos-cDNA fusion products, cysteines were engineered at the N- and C-termini of each of the leucine zippers, providing a covalent link of the cDNA gene product to the genetic instructions required for its production. Dissociation between phage and cDNA gene products was readily achieved using reducing agents. Phages displaying gene products covalently anchored on their surface via the modified leucine zippers can be selectively enriched 10(4)-10(6)-fold over nonspecific phages using antibodies. Thus, this cloning system allows rapid isolation of rare mRNA products from complex cDNA libraries by enrichment with appropriate ligands. This approach should allow the expression and cloning of dimeric proteins by cDNA shuffling.

Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus

An Aspergillus fumigatus extracellular alkaline protease (ALP) which is an enzyme of the subtilisin family is a potential virulent factor of the fungus. The gene encoding ALP was isolated from a genomic library made from DNA of an A. fumigatus isolate. The nucleotide sequence of this gene was compared to that of a cDNA encoding A. oryzae ALP and to that of a cDNA from A. fumigatus encoding the mature ALP protein. Mature A. fumigatus ALP contains 282 amino acids and is encoded by three exons. The pre-proenzyme has a leader sequence of 121 amino acids.

Diagnostic value of recombinant Aspergillus fumigatus allergen I/a for skin testing and serology

BACKGROUNDnWe report a clinical study comparing the recombinant Aspergillus fumigatus allergen I/a (rAsp f I/a) to two commercial A. fumigatus extracts in skin prick tests, intradermal tests, and serologic assays.nnnMETHODSnPatients with allergic bronchopulmonary aspergillosis and A. fumigatus-allergic patients with asthma, and control subjects, including allergic patients with asthma without allergy to A. fumigatus and healthy subjects, were investigated.nnnRESULTSnAll patients with allergic bronchopulmonary aspergillosis (n = 15) reacted to skin prick tests with the commercial extracts, and eight were sensitized to rAsp f I/a. Of 10 patients with well-characterized A. fumigatus-allergic asthma nine showed positive skin prick test results to at least one of the commercial extracts, and five reacted to rAsp f I/a. There was a strong correlation between skin test reactivity to rAsp f I/a and rAsp f I/a-specific serum IgE as determined by an antigen-specific ELISA. The healthy control subjects (n = 7) and allergic patients with asthma without A. fumigatus allergy (n = 6) did not react in skin prick and intradermal tests to rAsp f I/a, nor did they have detectable amounts of rAsp f I/a-specific IgE. In addition, patients with allergic bronchopulmonary aspergillosis showed significant elevated levels of rAsp f I/a-specific IgG4 and IgG1 but no significant differences in rAsp f I/a-specific serum IgA levels when compared with the healthy control subjects.nnnCONCLUSIONSnThe data show that rAsp f I/a is a major allergen with biologic relevance in some A. fumigatus-allergic individuals as evaluated by skin prick tests, intradermal tests, or serologic methods. Furthermore, no discrepancies were observed between skin test results and rAsp f I/a-specific IgE. Hence the correlation between rAsp f I/a skin test results and serologic data indicates the potential of recombinant allergens for clinical applications and diagnosis of allergies.

High-level expression in Escherichia coli and rapid purification of enzymatically active honey bee venom phospholipase A2☆

Bee venom phospholipase A2 (BV-PLA2) is a hydrolytic enzyme that specifically cleaves the sn-2 acyl bond of phospholipids at the lipid/water interface. The same enzyme is also believed to be responsible for some systemic anaphylactic reactions in bee venom sensitized individuals. To study the structure/function relationships of this enzyme and to define the molecular determinants responsible for its allergenic potential, a synthetic gene encoding the mature form of BV-PLA2 was expressed in Escherichia coli. This enzyme was produced as a fusion protein with a 6xHis-tag on its amino-terminus yielding 40-50 mg of fusion protein per 1 of culture after metal ion affinity chromatography. A kallikrein protease recognition site was engineered between the 6xHis-tag and the amino-terminus of the enzyme allowing isolation of the protein with its correct N-terminus. Recombinant affinity purified BV-PLA2 was refolded, purified to homogeneity, and cleaved with kallikrein, resulting in a final yield of 8-9 mg of active enzyme per 1 of culture. The enzymatic and immunological properties of the recombinant BV-PLA2 are identical to enzyme isolated from bee venom indicating a native-like folding of the protein.

Type I skin reactivity to native and recombinant phospholipase A2 from honeybee venom is similar

Phospholipase A2 is the major allergen in honeybee venom. Recombinant phospholipase A2 was produced in prokaryotes and tested for its biologic activity by intracutaneous skin testing with serial 10-fold dilutions in comparison with native and deglycosylated phospholipase A2 in patients allergic to bee venom. Linear regressions of the log of the wheal area versus the log of the allergen concentration were calculated for all allergens in each patient. The relative allergenic potency of the various preparations was analyzed by comparing the linear regressions. Native phospholipase A2 was about 10 times more potent than whole bee venom. None of 58 patients allergic to bee venom was missed by testing with native phospholipase A2 alone. This allergen and deglycosylated native phospholipase A2 resulted in similar skin reactions, indicating that the sugar residues were of little relevance for IgE-binding in the patients tested. Native phospholipase A2 also had relative potency similar to that of recombinant refolded phospholipase A2, whereas recombinant nonrefolded phospholipase A2 had almost no biologic activity in skin testing. These results demonstrate in vivo activity of the recombinant bee venom allergen phospholipase A2. Although correct refolding is a prerequisite for type I skin reactivity, glycosylation seems to be less important.

Antigen focusing by specific monomeric immunoglobulin E bound to CD23 on Epstein-Barr virus-transformed B cells

Monomeric IgE bound to the low-affinity receptor for IgE (FcERII-CD23) on EBV-transformed human B cells selectively enhances binding of antigen and therefore presentation to specific T-cell clones. To demonstrate the role of monomeric IgE in antigen focusing, we have made use of a system consisting of human T-cell clones specific for Der-P1 (major allergen of the Dermathophagoides pteronyssinus), Der-P1 coupled to NIP (Der-P1-NIP), and the commercially available chimeric (human-murine) monoclonal IgE antibodies with specificity for the hapten NIP. We have found that monomeric IgE binds to CD23 and remains detectable on the surface of the B cells for a period of at least 16 hours at 37 degrees C. Pulsing of these IgE-anti-NIP (1 microgram/ml) treated B cells for 1 hour at 37 degrees C with low amounts (10 ng/ml) of Der-P1-NIP antigen allows the B cells to stimulate Der-P1-specific T cells. Even with IgE concentrations as low as 20 ng/ml, which were not detectable by immunofluorescence, we were able to induce a significant T-cell response. Furthermore, ongoing specific T-cell-B-cell interactions were not inhibited by the presence of high concentrations of nonspecific IgE molecules (incubated with up to 25 micrograms/ml) on the surface of the B cells. Our data confirm the hypothesis that IgE, bound by either CD23 or the high-affinity receptor for IgE, potentiates the immune response. Therefore, IgE may be seen as the fourth general mechanism for antigen capture by (nonspecific) antigen-presenting cells.

Natural and recombinant enzymatically active or inactive bee venom phospholipase A 2 has the same potency to release histamine from basophils in patients with Hymenoptera allergy

BACKGROUNDnA complementary DNA encoding the major bee venom allergen phospholipase A2 (PLA) has been characterized recently. Recombinant PLA was produced in Escherichia coli and purified to apparent homogeneity. Natural PLA was compared with recombinant PLA in its ability to release histamine from blood basophils.nnnMETHODSnA synthetic gene encoding the mature form of PLA was expressed in E. coli, and the polypeptide was purified to homogeneity by affinity chromatography and refolded, yielding fully enzymatically active PLA. In addition, we have produced a genetically engineered enzymatically inactive variant by substitution of a single amino acid residue in the catalytic center. A standard histamine release assay was used to compare the potency of natural PLA with correctly folded enzymatically active and inactive recombinant PLA to release histamine from blood basophils of nine patients with bee venom allergy.nnnRESULTSnRecombinant enzymatically active PLA and purified natural protein were equally effective in releasing histamine from sensitized basophils. By comparing the histamine-releasing capacity of enzymatically active and inactive recombinant allergen, we further demonstrate that catalytic activity is not a requirement for allergenicity in the effector phase. Denaturation of natural PLA or incorrect folding of recombinant protein resulted in a total loss of allergenic potency.nnnCONCLUSIONnWe demonstrate the feasibility of producing native-like recombinant allergens with or without enzymatic activity. We also provide evidence for the requirement of correct three-dimensional structure of PLA to induce histamine release from basophils and thus evidence for its recognition by IgE.

Human monoclonal or polyclonal antibodies recognize predominantly discontinuous epitopes on bee venom phospholipase A2

BACKGROUNDnTwo hybridomas, which secrete human monoclonal antibodies of IgG4 isotype specific for the main bee venom antigen/allergen phospholipase A2, were generated. The antigenic determinants recognized by these antibodies were mapped and compared with the binding sites of murine monoclonal and human polyclonal antibodies raised against the same antigen.nnnMETHODSnTwo hybridomas were developed by fusing heteromyelomas to Epstein-Barr virus immortalized B cells obtained from beekeepers. The cloned hybridomas were stable and secreted up to 40 mg/L of antibody into the culture supernatant. Phospholipase A2 specificity of the human monoclonal antibodies was confirmed by binding and inhibition ELISA and by Western blot analysis. Epitope mapping on phospholipase A2 was done with the PEPSCAN method and ELISA techniques.nnnRESULTSnThe epitopes recognized by the human monoclonal antibodies were shown to be discontinuous and did not contain the sugar residue. Similar results were obtained with polyclonal antibodies of IgG4 isotype (from beekeepers) specific for phospholipase A2, which could also inhibit the binding of the human monoclonal antibodies to phospholipase A2. In contrast, antigen binding of the human monoclonal antibodies could not be inhibited by murine monoclonal antibodies against bee venom phospholipase A2.nnnCONCLUSIONSnThe data indicate that the human monoclonal antibodies obtained are representative of a part of the polyclonal immune response to phospholipase A2 from beekeepers and may allow a more precise analysis of the humoral immune response to phospholipase A2 that is associated with protection.

Rabbit single domain antibodies specific to protein C expressed in prokaryotes

VDJ genes were amplified by the polymerase chain reaction from mRNA isolated from peripheral blood B cells of rabbits immunized with protein C. The amplified genes were cloned into a lambda phage expression vector and packaged. A library of 6 x 10(5) recombinant phages was screened with labelled protein C and 30 positive clones were found. Three of them were plaque purified and the affinity of the single domain antibodies to the antigen determined to be 10(6)-10(7) l M-1. The data indicate the feasibility of generating single domain antibody, specific to protein antigen, from rabbit.

Lysine Residues in Bee Venom Phospholipase A2 Are Important for Binding to Human Monoclonal or Polyclonal Antibodies of the lgG4 Isotope

Discontinuous antigenic sites on bee venom phospholipase A2 (PLA) have been mapped using human monoclonal antibodies or human polyclonal serum antibodies (hpAbs) of the IgG4 isotype from beekeepers or of the IgE isotype from individuals allergic to PLA. Lysine residues of PLA have been specifically modified by acetylation or acylation by treatment with citraconic anhydride of their epsilon-amino groups to analyze their role in antigen-antibody binding. After the modifications, the binding of PLA to the human monoclonal antibodies is lost, whereas the binding to IgG4 hpAbs is significantly decreased. In contrast, the effect on the binding of PLA to IgE hpAbs appears to be more heterogeneous. The data indicate the importance of lysine residues as being part of B cell epitopes in PLA-specific antibodies of the IgG4 isotype, but less so for those of the IgE isotype.