Takaomi Yasuhara

Biologically active recombinant forms of a major house dust mite group 1 allergen Der f 1 with full activities of both cysteine protease and IgE binding.

Background Group 1 allergens from mite faeces, Der f 1 and Der p 1, are the most significant in‐door allergens. Therefore, they are the most important component in the standardization of house dust mite extract for diagnosis and allergen‐specific immunotherapy (AIT). Although their cDNAs have been cloned, efforts to prepare biologically active recombinant forms in expression systems using bacteria or yeast have failed.

Allelic imbalance in chromosome band 18q21 and SMAD4 mutations in ovarian cancers

Recently, three candidate tumor suppressor genes, SMAD2 (MADR2/JV18–1), SMAD4 (DPC4), and DCC, were identified in chromosome band 18q21. We examined allelic imbalance (AI) in 18q21 using six polymorphic microsatellite markers in 38 primary ovarian cancers and four ovarian borderline tumors. AI at one or more loci was detected in 15 of 37 (41%) informative ovarian cancers and in none of the four borderline tumors. Frequent AI was detected at the D18S46 (31%) and D18S474 (36%) loci, which were adjacent to the SMAD4 gene, and at the D18S69 (33%) locus, which was telomeric to the DCC gene. Therefore, we searched for mutations of the SMAD4 gene in 42 primary tumors and eight cell lines by PCR‐SSCP and sequencing analyses. Missense mutations were detected in two ovarian tumors and three ovarian cancer cell lines, whereas silent mutation was detected in a primary ovarian cancer. These results suggest that there are at least two tumor suppressor genes on chromosome arm 18q and that SMAD4 is of importance in ovarian tumorigenesis. Genes Chromosomes Cancer 24:264–271, 1999.

Production of enzymatically and immunologically active Der f 1 in Escherichia coli

Der f 1 is a major house dust mite allergen belonging to the cysteine protease family. Because of the great demand for clinical and research use of this allergen, much effort to establish an efficient method of preparing purified Der f 1 has been made. We constructed an isopropyl-β-D-thiogalactopyranoside-inducible expression plasmid to produce the pro-form of Der f 1 in Escherichia coli. The recombinant product was accumulated as insoluble inclusion bodies in cells. The solubilized inclusion bodies were found to be successfully renatured by two-step gel filtration chromatography. About 70 mg of pro Der f 1 were properly refolded by this method from 1 liter of culture. Acid treatment of the renatured pro Der f 1 resulted in the autocatalytic removal of the pro-sequence. The obtained mature form of Der f 1 bound IgE in patient sera and induced the release of histamine from peripheral blood leukocytes equally to native Der f 1. Furthermore, mature Der f 1 obtained by this method had identical protease activity with native Der f 1. We also discussed the contribution of the pro-sequence and the sugar chain of Der f 1 to its antigenic and enzymatic activity. This is the first report to produce an active mature form of recombinant Der f 1 in E. coli.

Effects of site-directed mutagenesis in the cysteine residues and the N-glycosylation motif in recombinant Der f 1 on secretion and protease activity.

Background: The group 1 allergens from mite feces, which belong to the papain-like cysteine protease family, are the most significant in-door allergens. In this study, we analyzed the contribution of the cysteine residues and N-glycosylation in Der f 1, the group 1 allergen from Dermatophagoides farinae, to secretion and maturation by using systems for expression of recombinant Der f 1 (rDer f 1). Methods: The rDer f 1 and its mutants were expressed in yeast Pichia pastoris and insect SF9 cells. Secretion of their proforms was checked by SDS-PAGE or immunoblotting. Protease activities of the secreted proform of a mutant and the mature form were compared with that of native Der f 1. Results: The proform of a mutant Der f 1, pro-N53Q, whose consensus motif for N-glycosylation was disrupted, was not secreted in insect SF9 cells although secreted in P. pastoris. Indirect evidence was obtained to support the disulfide bond formation between Cys4 and Cys118, which were not conserved in papain. A mutant for Cys35 in the catalytic site of the cysteine protease, pro-C35S/N53Q, was secreted, but the other mutants for cysteines concerning intramolecular disulfide bonds were not secreted in P. pastoris. The prosequence of pro-C35S/N53Q was removed by an in vitro activation process. The mature C35S/N53Q showed low protease activity. Conclusion: N-glycosylation is essential for secretion in insect SF9 cells but not in P. pastoris. Disulfide bonds are essential for secretion in P. pastoris. A mutation in the catalytic site, C35S, is not completely critical to removal of the prosequence and protease activity. The findings are useful for future design of recombinant products for application in immunotherapy.

Direct expression of Der f2, a major house dust mite allergen, in Escherichia coli.

Der f2 protein in a highly antigenic form was directly expressed in bacteria. Plasmid pFLU11 derived from pKK233-2 was designed to express methionyl-Der f2 under the control of the trc promoter and it has the replication origin of pUC118 instead of its original to increase the copy number. This expression plasmid directed the synthesis of recombinant Der f2 (rDer f2) protein in an insoluble form of inclusion bodies in Escherichia coli cells. The high copy number plasmid pFLU11 conferred the efficient production of the Der f2 protein in E. coli, when compared to a nonchanged origin material. rDer f2 inclusion bodies were easily solubilized in urea and renatured by dialysis to assume the active form. The rDer f2 protein was purified by means of anion exchange and gel filtration chromatography. This expression system yielded about 10 mg of purified rDer f2 protein from the 1L culture. Purified rDer f2 protein reacted with IgE from patient sera almost identically to the native Der f2 in the RAST enzyme immunoassay and skin prick test.

Induction of plant disease resistance upon treatment with yeast cell wall extract

It has been reported that treatment with yeast cell wall extract (YCWE) induces PDF1 and PR-1 gene expression; these transcripts are important markers of plant disease resistance, though the detailed signaling mechanisms that induce these defense responses are still unknown. In this report, we found that YCWE treatment triggered rice cell suspension cultures to accumulate phenylalanine (Phe), cis-12-oxo-phytodienoic acid (OPDA), 12-hydroxyjasmonoyle isoleucine (12OHJA-Ile), and azelaic acid (AzA). YCWE treatment also reduced endogenous triacylglycerol (TG) content. The addition of 13C-uniform-labeled oleic, linoleic and linolenic acids to the rice cell suspension cultures gave rise to 13C-uniform-labeled AzA. It was also found that YCWE treatment for Arabidopsis thaliana resulted in accumulations of OPDA, AzA, Phe, and camalexin together with enhanced resistance against Botrytis cinerea infection. This suggested that YCWE treatment upon plants may activate JA and AzA signaling systems to induce plant disease resistance. Yeast cell wall extract (YCWE) treatment induces plant defense response.