High-Level Cell-Free Synthesis Yields of Proteins Containing Site-Specific Non-Natural Amino Acids
High-Level Cell-Free Synthesis Yields of Proteins Containing Site-Specific Non-Natural Amino Acids
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DOI：
10.1002/bit.22070
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发表时间：
2009-02-01
影响因子：
3.8
通讯作者：
Swartz, James R.
中科院分区：
文献类型：
--
作者：
Goerke, Aaron R.;Swartz, James R.
我们描述了一种基于大肠杆菌的无细胞系统，用于生产具有非天然氨基酸(NnAA)结合位点特异性(修饰蛋白)的蛋白质。以突变的简氏甲烷球菌酪氨酰-tRNA合成酶(MTyrRS)和tRNatyr对为正交元。随着合成酶加入量的增加(200-300微克/毫升)，mTyrRS发生了蛋白降解，修饰蛋白产量提高。通过将总蛋白水平提高到20毫克蛋白质/毫升和有效囊泡表面积增加到0.5m(2)/毫升，产品产量也得到了提高。这种新的基于大肠杆菌的无细胞程序产生了高达400微克/毫升的eCAT109pAz、660微克/毫升的eDHFR10pAz和210微克/毫升的mDHFR31pAz，并将对叠氮-L-苯丙氨酸(PAZ)结合在琥珀无意义密码子上。O-甲基-L-酪氨酸和对-乙酰基-L-苯丙氨酸通过类似的方案合成。证实了通过无细胞系统掺入nnAA所需的特异性。此外，修饰的蛋白质对铜(I)催化的(3+2)环加成反应具有酶活性(点击化学)。
We describe an E. coli-based cell-free system for the production of proteins with a non-natural amino acid (nnAA) incorporated site-specifically (modified protein). The mutant Methanococcus jannaschii tyrosyl-tRNA synthetase (mTyrRS) and tRNATyr pair were used as orthogonal elements. The mTyrRS experienced proteolysis and modified protein yields improved with higher synthetase addition (200-300 mu g/mL). Product yields were also improved by increasing levels of total protein to 20 mg protein/mL and available vesicle surface area to 0.5 m(2)/mL This new E. coli-based cell-free procedure produced up to 400 mu g/mL, of eCAT109pAz, 660 mu g/mL of eDHFR10pAz, and 210 mu g/mL of mDHFR31pAz with p-azido-L-phenylalanine (pAz) incorporated site-specifically at the amber nonsense codon. O-methyl-L-tyrosine and p-acetyl-L-phenylalanine were incorporated by similar protocols. The desired specificity for incorporation of the nnAA by the cell-free system was confirmed. Additionally, the modified proteins were enzymatically active and reactive for copper (I)-catalyzed (3 + 2) cycloadditions (click chemistry).