Adult male and female German cockroaches were reared for 24℃ on an artificial diet, and given free access to water.

A B. germanica cDNA library was constructed with Uni-ZAPTM XR expression vector (Stratagene, USA). In brief, the total RNA was isolated from 3 g of the midguts of adult B. germanica specimens. Following phenol extraction and ethanol precipitation, poly(A+) RNA was purified with a Stratagene Poly(A) Quick mRNA Isolation Kit, in accordance with the manufacturer's instructions. First-strand cDNA synthesis of the isolated poly(A+) RNA was then conducted in 50 µl reaction volumes, using 50 units of MMLV-reverse transcriptase at 37℃ for 60 minutes. cDNA synthesis was primed using 5 µg oligo dT18. Second-strand synthesis was then conducted using RNase H and DNA polymerase I. After blunting the cDNA termini, we conducted EcoR I adaptor ligation and EcoR I phosphorylation. The region of the gel harboring the DNA molecules with lengths of less than 400 bp was then removed via Sepharose CL-2B gel filtration. The purified cDNA was ligated using dephosphorylated EcoR I Uni-ZAPTM XR vector arms, according to the manufacturer's instructions, then incubated using in vitro packaging extracts (Stratagene).

Ten serum samples were obtained from patients manifesting symptoms of perennial rhinitis, asthma, and eczema. These patients had been diagnosed with allergies, via RAST and skin prick tests. A set of control sera was acquired from 5 individuals, who were found not to be allergic to cockroaches, or to any other common allergens. The collection of sera for use in these studies was approved by the Human Subjects Investigation Committee of Kosin University. The library was screened using 1:10, 1:5, or 1:2 serum dilutions (pre-absorbed with Escherichia coli lysates) pooled from patients who had proven sensitive to B. germanica. Positive plaques were detected using alkaline phosphatase-labeled goat anti-human IgE (1:500). The p-Bluescript phagemid harboring the cloned cDNA insert of the positive plaque was then excised from the Lambda Zap II vector, and was plated with fresh E. coli cells, in order to foster the formation of colonies (ExAssit/SOLR system: Stratagene). Doublestranded cDNA was then isolated and sequenced (Macrogen, DNA Sequencing Service, Seoul, Korea).

The partial trypsin cDNA clone was subjected to plaque hybridization in order to generate full-length cDNA, in accordance with the manufacturer's instructions (Stratagene).

Protein and nucleotide sequences were compared with the data from the nonredundant GenBank, EMBL, SwissProt, DBJ, GenPept, PDB, and PIR databases, using FASTA (GCG, Oxford Molecular Group, Inc). The sequence alignments were assessed using the GCG program.

10 µg of B. germanica genomic DNA was digested using 5 Units of EcoR I, Xho I, and Hind III (Boehringer Mannheim, Germany). The DNA digests were separated on 0.8% agarose gel, then transferred to Hybond-N⁺ nylon membranes, according to the manufacturer's instructions (Amersham, USA). The membranes were blocked and hybridized with [³²p]cDNAlabeled probes prepared with a randomly primed DNA labeling kit (Boehringer Mannheim, Germany). Prehybridization and hybridization were conducted at 60℃ with [³²p]-labeled cDNA. After washing, the filters were air-dried, and exposed to X-ray film.

Total RNA was extracted from the midgut tissues of adult cockroaches, as was described above. The RNA was separated on denaturing 1.0% agarose gel harboring 6.7% formaldehyde, then transferred to Hybond-N⁺ nylon membranes (Amersham, USA). The probe was constructed with the full-length trypsin gene, using the random prime labeling method (Boehringer Mannheim, Germany), and we conducted standard Northern hybridization analysis.

Trypsin-encoding cDNA was cloned into the pGEXKG expression vector and expressed in E. coli BL21 cells (DE3). Trypsin-encoded cDNA was then transferred to pGEX-KG as a BamH I-EcoR I fragment. Competent E. coli BL21 (DE3) cells were allowed to grow to an O.D. 600 of 0.6 in Luria-Bertani broth supplemented with 200 µg/ml of ampicillin. Isopropylthio-β-galactoside was then added to a final concentration of 0.6 mM, and incubation proceeded for an additional 3 hours. An uninduced control culture was also prepared without IPTG. The cells were harvested via centrifugation, and the pellets were resuspended in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1 mM EDTA, harboring 100 µg/ml of lysozymes, then incubated for 15 minutes at 37℃. After mild sonication (six pulses of 10 seconds each), the recombinant trypsin, which was Glutathione S-transferase fusion protein in the E. coli, was induced 0.3 mM IPTG at 37℃, 3 h. The harvested cells were washed in cold PBS, after which we added ice-cold lysis buffer (25 mM HEPES-KOH, pH 7.9, 100 mM KCl, 10% glycerol, 2 mM EDTA, 2 mM DTT) containing 200 µg/ml of lysozymes. Both the induced and control bacteria were then pelleted and frozen at -20℃ until use. After sonication, the centrifuged supernatants were added to 2 ml glutathione-affinity resin, and cultured at 4℃, for 30 minutes. The supernatant was again washed with 10 volume thrombin digestion buffer (50 mM Tris-HCl, pH 8.3, 100 mM NaCl, 2.5 mM CaCl₂) and 20 µg thrombin was added (Sigma, USA). After protein retrieval, the proteins were further concentrated using Centriprep 10 and Centricon 10 (Millipore USA). The concentrated proteins were then diluted using a 4 volume of equilibration buffer (50 mM HEPES, pH 7.6, 10% glycerol, 1 mM EDTA, 1 mM DTT). The released fractions were collected and applied to a gel filtration column, in order to purify the trypsin to homogeneity.

The IPTG-induced and uninduced BL21/trypsin bacterial lysates were boiled in SDS sample buffer supplemented with β-mercaptoethanol. The constituent proteins were then fractionated via SDSPAGE, and were transferred to nitrocellulose membranes (pore size, 0.45 µm: Millipore USA). After being electrophoretically transferred, the membranes were blocked for 1 hour with 5% skim milk in PBST. The blots were washed 3 times with PBST, and then stored at 4℃ in the sera of the cockroach allergy patients overnight. After 3 times washings with PBST, the membranes were reincubated with horseradish peroxidase-labelled goat anti-human IgE (Dako, Denmark) diluted to 1:500 in PBST-BSA for 2 hours at room temperature. The membranes were then developed using DAB (Sigma, diaminobenzidine 6 mg, 50 mM tris-HCl, pH 6.7 9 ml, 0.3% nickel chloride 1 ml, hydrogen peroxide 10 µl).

After the construction of the cDNA library, the mean size of the inserts was determined to be 0.3-2.0 Kb, and the titer of the cDNA library was measured at 10¹² pfu/ml.

The immunoscreening of the cDNA library with the German cockroach allergy patients revealed a partial trypsin clone. In order to generate the full-length cDNA sequence of this trypsin, we conducted a plaque hybridization of the B. germanica cDNA library, using the cDNA fragment as a probe. The largest insert exhibited a single open reading frame of 863 bp, which predicted the formation of a protein which was composed of 257 amino acid residues (Fig. 1, bgtryp-1). At the 3' end of bgtryp-1, we noted a translation termination codon, followed by 45 nucleotides of a presumably untranslated sequence, and a poly(A) tail (GenBank accession number DQ149980). On the basis of the BLASTP homology searches, the deduced amino acid sequences of the cloned genes were clearly determined to belong to a trypsin-like protease. bgtryp-1 exhibited 40% and 35-37% similarity with insect trypsin genes and mammalian trypsin genes, respectively (Fig. 2). It was also 42-57% similar in sequence to several mite serine protease allergens, including Der f 3 (Dermatophagoides farinae), Eur m 3 (Euroglyphus maynei), Der p 3 (Dermatophagoides pteronyssinus), and the fully-conserved catalytic domain, GDSGGPLV (Fig. 3).

The genomic DNA from the cockroach was separated and digested using 3 kinds of enzymes, all of which lacked target sites in the gene. A cDNA probe of approximately 500 bp was used in our Southern blot hybridization (Fig. 4). The observation of only one band indicated that this clone was a single-copy gene. The Northern blot evidenced a 0.9 kb transcript (Fig, 5).

Expression of the recombinant proteins in E. coli BL21, resulted in high level of B. germanica trypsin protein (Bgtryp-1) visualized by Coomassie Blue staining of whole bacterial lysates separated by SDSPAGE. Thick band of expected sizes of fusion protein with GST (glutathione-S-transferase) was calculated as 49 kDa (Fig. 6A lane 4) and 35 kDa when the signal was detached by thrombin (Fig. 6A lane 2 and Fig. 6B). The proteins expressed were contained in soluble fraction of the bacterial lysates. After purification of the fusion proteins, bands were confirmed to be located at their respective expected sizes. The recombinant protein detached signal reacted with sera positive to cockroach allergen at 35 kDa level (Fig. 6C).