Young Travis S, Ahmad Insha, Brock Ansgar, Schultz Peter G
Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, USA.
Biochemistry. 2009 Mar 31;48(12):2643-53. doi: 10.1021/bi802178k.
To increase the utility of protein mutagenesis with unnatural amino acids, a recombinant expression system in the methylotrophic yeast Pichia pastoris was developed. Aminoacyl-tRNA synthetase/suppressor tRNA (aaRS/tRNA(CUA)) pairs previously evolved in Saccharomyces cerevisiae to be specific for unnatural amino acids were inserted between eukaryotic transcriptional control elements and stably incorporated into the P. pastoris genome. Both the Escherichia coli tyrosyl- and leucyl-RS/tRNA(CUA) pairs were shown to be orthogonal in P. pastoris and used to incorporate eight unnatural amino acids in response to an amber codon with high yields and fidelities. In one example, we show that a recombinant human serum albumin mutant containing a keto amino acid (p-acetylphenylalanine) can be efficiently expressed in this system and selectively conjugated via oxime ligation to a therapeutic peptide mimetic containing an permittivity-(2-(aminooxy)acetyl)-L-lysine residue. Moreover, unnatural amino acid expression in the methylotrophic host was systematically optimized by modulation of aaRS levels to express mutant human serum albumin in excess of 150 mg/L in shake flasks, more than an order of magnitude better than that reported in S. cerevisiae. This methodology should allow the production of high yields of complex proteins containing unnatural amino acids whose expression is not practical in existing systems.
为了提高利用非天然氨基酸进行蛋白质诱变的效用,开发了一种在甲基营养型酵母毕赤酵母中的重组表达系统。先前在酿酒酵母中进化出的对非天然氨基酸具有特异性的氨酰 - tRNA合成酶/抑制tRNA(aaRS/tRNA(CUA))对,被插入到真核转录控制元件之间,并稳定整合到毕赤酵母基因组中。大肠杆菌的酪氨酰 - 和亮氨酰 - RS/tRNA(CUA)对在毕赤酵母中均显示为正交,并用于以高产率和保真度响应琥珀密码子掺入八种非天然氨基酸。在一个实例中,我们表明含有酮氨基酸(对乙酰基苯丙氨酸)的重组人血清白蛋白突变体可以在该系统中有效表达,并通过肟连接选择性地与含有允许性 - (2 - (氨基氧基)乙酰基)-L - 赖氨酸残基的治疗性肽模拟物偶联。此外,通过调节aaRS水平系统地优化了甲基营养型宿主中的非天然氨基酸表达,以在摇瓶中表达超过150 mg/L的突变型人血清白蛋白,比酿酒酵母中报道的产量高出一个数量级以上。这种方法应该能够高产率地生产含有非天然氨基酸的复杂蛋白质,而这些蛋白质在现有系统中表达是不切实际的。