Removal of N-terminal methionine from recombinant proteins by engineered E-coli methionine aminopeptidase
Removal of N-terminal methionine from recombinant proteins by engineered E-coli methionine aminopeptidase
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DOI：
10.1110/ps.04679104
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发表时间：
2004-07-01
期刊：
影响因子：
8
通讯作者：
Chang, ST
中科院分区：
文献类型：
--
作者：
Liao, YD;Jeng, JC;Chang, ST
甲硫氨酸氨基肽酶 (MetAP) 去除 N 端翻译起始子 Met 对于蛋白质的功能和稳定性通常至关重要。在MetAP的晶体结构和序列比对的基础上，我们通过底物结合口袋中三个残基Y168G、M206T、Q233G的突变设计了大肠杆菌MetAP。我们的工程化 MetAP 能够去除大体积或酸性倒数第二个残基（例如 Met、His、Asp、Asn、Glu、Gln、Leu、Ile、Tyr 和 Trp）以及小残基中的 Met。如果倒数第二个残基（Met之后的第三个残基）较小，则进一步除去倒数第二个残基（Met之后的第二个残基）。通过通过相同或单独的载体在大肠杆菌中共表达工程化的 MetAP，我们成功地产生了具有先天 N 末端的重组蛋白，例如 onconase，一种来自青蛙 Rana pipiens 的抗肿瘤核糖核酸酶。重组 Onconase 的 N 端焦谷氨酸对其结构完整性、催化活性和细胞毒性至关重要。根据蛋白质数据库中的 N 端序列信息，85%-90% 的重组蛋白应由我们的工程化 MetAP 以真实形式产生。
The removal of N-terminal translation initiator Met by methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins. On the basis of crystal structure and sequence alignment of MetAPs, we have engineered Escherichia coli MetAP by the mutation of three residues, Y168G, M206T, Q233G, in the substrate-binding pocket. Our engineered MetAPs are able to remove the Met from bulky or acidic penultimate residues, such as Met, His, Asp, Asn, Glu, Gln, Leu, Ile, Tyr, and Trp, as well as from small residues. The penultimate residue, the second residue after Met, was further removed if the antepenultimate residue, the third residue after Met, was small. By the coexpression of engineered MetAP in E. coli through the same or a separate vector, we have successfully produced recombinant proteins possessing an innate N terminus, such as onconase, an antitumor ribonuclease from the frog Rana pipiens. The N-terminal pyroglutamate of recombinant onconase is critical for its structural integrity, catalytic activity, and cytotoxicity. On the basis of N-terminal sequence information in the protein database, 85%-90% of recombinant proteins should be produced in authentic form by our engineered MetAPs.