Cloning and Expression of Aspartic Proteinase cDNA from Sweet Potato Storage Roots

甘藷塊根天門冬胺酸蛋白酶cDNA之選殖和表現

黃冠中(Guan-Jhong Huang) ； 黃世勳(Shyh-Shyun Huang) ； 陳顯榮(Hsien-Jung Chen) ； 張永勳(Yuan-Shiun Chang) ； 張淑貞(Shu-Jen Chang) ； 謝伯舟(Heng-Yuan Chang) ； 張恆源(Po-Chow Hsieh) ； 張曼釗(Man-Jau Chang) ； 林穎志(Ying-Chih Lin) ； 林耀輝(Yaw-Huei Lin)

《Botanical Studies》 50卷2期 (2009/04) Pp. 149-158

摘要

利用差異表現法(differential display)分離甘藷(Ipomoea batatas [L.] Lam. 'Tainong 57')塊根之天門冬胺酸蛋白酶(Aspartic proteinases; EC, 3.4.23; SPAP)。SPAP之開放轉譯架構(open reading frame)轉譯成508個胺基酸，預測其分子量為55,006 Da (pI 4.91)。SPAP基因和甘藷老化葉中之天門冬胺酸蛋白酶基因(SPAPSL)在DNA和蛋白質序列比對時，具有81%和78%相似性。SPAP胺基酸序列和其他之AP胺基酸序列差異在於訊息胜肽和前胜肽的部分。胺基酸序列含有天門冬胺酸蛋白酶基因保守性特徵，包括plant specific insert (PSI)和兩個活性區之天門冬胺酸基。藉由北方點墨法檢視SPAP在甘藷表現量的多寡發現SPAP之轉錄專一性的表現在塊根中。在大腸桿菌(M15)中大量表現重組蛋白質SPAP，然後利用鎳離子螯合之親和性管柱純化。當22 kDa甘藷胰蛋白酶抑制因子(trypsin inhibitor, TI)被dithiothreitol(DTT)完全還原後，可被具活性之重組蛋白質SPAP分解。但是當TI被NTS(NADP/thioredoxin system)部分還原後，卻不能夠被具活性之重組蛋白質SPAP分解。這些結果顯示當22 kDa SPTI由NTS (NADP/thioredoxin system)部分還原後，先由專一性之半胱胺酸蛋白酶進行初步之降解；接著再由本文所描述之SPAP進行後續的水解過程。

關鍵字

天門冬胺酸蛋白酶 ； 基因表現 ； 重組蛋白質 ； 甘藷 ； 胰蛋白酶抑制因子

並列摘要

Aspartic proteinases (EC, 3.4.23) cDNA clone (SPAP) of sweet potato (Ipomoea batatas (L.) Lam. 'Tainong 57') storage roots were isolated by differential display. The open reading frame in this cDNA encodes a pre-pro-protein of 508 amino acids with a predicted molecular mass of 55,006 Da (pI 4.91). The SPAP gene shares 81% and 78% homology on the level of nucleotides and amino acids, respectively, with an aspartic proteinase cDNA of sweet potato senescent leaves (SPAPSL). SPAP amino acid sequence was different from other AP sequences in signal and propeptide portions. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant specific insert (PSI) and two active site aspartic acid residues. Examination of the expression patterns in sweet potato by northern blot analyses revealed that the transcripts of SPAP were specifically induced in the storage roots. Recombinant SPAP overproduced in E. coli (M15) was purified by Ni(superscript 2+)-chelated affinity chromatography. Active recombinant SPAP was able to digest the 22 kDa sweet potato trypsin inhibitor (TI) when the latter was reduced by dithiothreitol (DTT). SPAP could not degrade bands of reduced TI when NTS (NADP/thioredoxin system) was used to reduce TI. These results suggest that SWAP has an in vivo proteolytic function of processing storage SPTI after its being degraded initially by a specific cysteine proteinase.

並列關鍵字

Aspartic proteinase ； cDNA sequence ； Recombinant protein ； Sweet potato ； Trypsin inhibitor