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研究生:陳盈志
研究生(外文):Chen Ying-Chih
論文名稱:人類胎盤型鹼性磷酯■在原核與真核細胞表現之研究
論文名稱(外文):Expression of human placental alkaline phosphatase in prokaryotic and eukaryotic cells
指導教授:張自忠;張固剛
指導教授(外文):Chang Tsu-Chung;Chang Gu-Gang
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:1996
畢業學年度:84
語文別:中文
論文頁數:66
中文關鍵詞:鹼性磷酯■蛋白質表現突變
外文關鍵詞:Alkaline phosphataseProtein expressionMutagenesis
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人類胎盤型鹼性磷酯■為一種膜蛋白,可催化磷酯化合物的水解反應,活
性較相對應細菌鹼性磷酯■高出20-30倍。大腸桿菌與哺乳類動物之鹼性
磷酯■胺基酸序列約有30%完全相同,比較兩者活性中心之胺基酸序列,
發現大腸桿菌鹼性磷酯■胺基酸序列153及328分別為天門冬胺酸及離胺酸
,而在哺乳類動物鹼性磷酯■中,則兩個胺基酸序列均為組織胺酸。先前
研究顯示大腸桿菌鹼性磷酯■胺基酸序列153可能是造成人類鹼性磷酯■
與細菌鹼性磷酯■活性差異的原因之一。本實驗首先構築原核細胞表現質
體,企圖以大腸桿菌於IPTG及rifampicin的調控下,表現人類胎盤型鹼性
磷酯■。由於大腸桿菌所表現之人類胎盤型鹼性磷酯■大多於菌體內形成
不可溶之涵體,無顯著之活性。我們轉而構築胎盤型鹼性磷酯■之真核細
胞表現質體,以細胞轉染之方式,使細胞表現人類胎盤型鹼性磷酯■。為
了瞭解組織胺酸在此■活性之重要性,我們以DNA定位突變方式將哺乳類
胎盤型鹼性磷酯■胺基酸序列153由組織胺酸變為天門冬胺酸或丙胺酸,
所得之突變型胎盤型鹼性磷酯■活性較野生型胎盤型鹼性磷酯■活性分別
降低13倍與400倍,顯示哺乳類胎盤型鹼性磷酯■胺基酸序列153之組織胺
酸對酵素活性有著相當程度的重要性。此外,本研究也顯示細胞表現鹼性
磷酯■之活性高低與細胞生長速率並無直接關係。

Alkaline phosphatase is a GPI-anchored integral membrane
protein catalyzing the hydrolysis of phosphoester linkage.
Mammalian alkaline phosphatases are 20-30 folds more active
than the corresponding bacterial enzyme. Earlier studies with
the E. coli enzyme suggested that the amino acid position 153
is one of the two noticeable sites that are responsible for the
difference in catalytic properties between the bacterial and
human alkaline phosphatase. In order to explore the role of
His-153 residue in the human enzyme, we replaced the His-153
with Asp or Ala by site-directed mutagenesis in this study. The
cDNA of this enzyme was subcloned into a series of expression
vectors, in an attempt to overexpress the human placental
alkaline phosphatase. At first, the prokaryotic expression
system was used. However, probably due to misfolding or lack of
post-translational modifications in E. coli, the majority of
the bacterially over- expressed enzyme molecules was inactive
and accumulated in in- soluble inclusion bodies. The tight
controlled eukaryotic ex- pression system was then employed to
express the enzyme in cul- tured human cells. The enzyme
expression plasmid was transient- ly transfected and the
expressed enzymes were partial purified for further studies.
Kinetic studies indicated that mutations of His-153 to Asp and
Ala reduced the enzyme activity 13 to 400 folds respectively.
The His residue at position 153 of human alkaline phosphatase
therefore appears to play an essential role in the enhanced
catalytic function of this enzyme in human cells. In addition,
using stably transfected cell lines express- ing various level
of placental alkaline phosphatase, we found that the level of
expressed placental alkaline phosphatase activity was not
related to the proliferation rate of MCF-7 cells.

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