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標題: Cloning of leucine aminopeptidase gene from Aspergillus oryzae
Aspergillus oryzae leucine aminopeptidase基因的選殖
作者: 趙秀慧
Chao, Shiou Huei
關鍵字: leucine aminopeptidase
亮胺酸N 端切位酉每
Aspergillus oryzae
出版社: 分子生物學研究所
摘要: Aspergillus oryzae is widely used in the fermentation industry. This fungus can to secrete many types and high level of proteases to decompose nutrient sources. Leucine aminopeptidase (LAP) may participate in protein degradation. A. oryzae LAP is commercialized, however, its gene structure and physiological role has never been reported. To obtain a lap gene fragment as probe, LAP was purified from commercial Aspergillus sojae proteases using FPLC and RP-HPLC. The purified LAP was digested with endopeptidase Glu-C or lyslyendopeptidase. Partial LAP fragments were sequenced and back-translated to degenerate primers. A 700bp fragment was amplified by PCR using A. oryzae cDNA as template. The lap genes were then isolated from A. oryzae genomic and cDNA libraries using 700 bp fagment as probe. A. oryzae lap gene comprises 1,320 bp with 3 introns and encodes a protein consisting of 377 amino acids ( 40,506 Da). The deduced amino acids shows only 33% identity to V. proteolyticus lap. Comparison of the protein sequencing from A. sojae LAP and the deduced amino acid sequence from A. oryzae lap revealed a signal peptide with 77 amino acids in A. oryzae LAP. The mature protein has a calculated molecular mass of 36,280 Da which almost matches the molecular mass of 35 kDa determined from SDS-PAGE. A TATA box was identified at position -120 from the ATG, and a CAAT box was found at position -286. A specific region, -88 to -69, may be related to pH regulation. A poly (A) adding site was found in the 3'' region. Three introns are 59, 74, and 56 bp in size and located at 155-213, 640-713 and 1031-1086, respectively. Except the intron which is the nearest to start codon, others obeys the GT-AG rule. Two putative N-glycosylation sites could be identified in Asn-87 and Asn-288 of A. oryzae lap.
Aspergillus oryzae是醱酵工業上經常使用的菌種。此種真菌能分泌多種蛋白酉每,以分解環境中的物質,作為營養來源。Leucine aminopeptidase (LAP) 可能也是扮演相同的角色。在市面上已經有來自A. oryzae 的LAP商品,但是目前仍未有lap基因序列及其生理意義的相關文獻被發表。 為取得lap基因片斷做為探針,將市售的Aspergillus sojae蛋白 酉每,經FPLC及RP-HPLC純化出LAP,再分別以endopeptidase Glu-C及lysylendopeptidase處理後,回收純化胜月太。經氨基酸定序分析,將定序所得的氨基酸序列,設計成degenerate primer,以A. oryzae cDNA為模板 (template),利用聚合酉每 鏈鎖反應 (PCR),複製出700 bp的片段。以此片段作為探針 (probe),由A. oryzae染色體基因庫及cDNA基因庫中,分別選殖出lap基因。 A. oryzae lap基因的ORF全長為1,320 bp,帶有3個introns,可轉譯出377個氨基酸(40,506 Da)。轉譯出的氨基酸序列,與V. proteolyticus lap基因具有33 % 的相似性。將A. sojae LAP蛋白之N端氨酸基序列與A. oryzae lap基因比對,顯示在A. oryzae lap基因中,具有由77個氨基酸組成的訊號胜月太 (signal peptide),俾將LAP外泌到胞外。分泌到胞外的LAP,分子量為32,680 Da,與利用SDS-PAGE上所測得的分子量 (35 kDa) 接近。在距ATG -120 ~ -114,有一TATA box,另有CAAT box位於-286 ~ -283。在-88 ~ -69位置,有一個特殊的DNA序列,可能是pH的調控區域。在ORF的3''端,有poly (A) adding site。在ORF內的3個introns,大小分別為59、74及56 bp,距離ATG 155-213、640-713 及1031-1086的位置。除了最靠近轉錄起始點的intron外,其餘兩個introns的結構,皆符合GT-AG的原則。lap基因的Asn-87與Asn-288,可能是會被醣化的位置。
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