title: Ligation of expressed protein α-hydrazides via genetic incorporation of an α-hydroxy acid.
abstract: Expressed protein ligation bridges the gap between synthetic peptides and recombinant proteins and thereby significantly increases the size and complexity of chemically synthesized proteins. Although the intein-based expressed protein ligation method has been extensively used in this regard, the development of new expressed protein ligation methods may improve the flexibility and power of protein semisynthesis. In this study a new alternative version of expressed protein ligation is developed by combining the recently developed technologies of hydrazide-based peptide ligation and genetic code expansion. Compared to the previous intein-based expressed protein ligation method, the new method does not require the use of protein splicing technology and generates recombinant protein α-hydrazides as ligation intermediates that are more chemically stable than protein α-thioesters. Furthermore, the use of an evolved mutant pyrrolysyl-tRNA synthetase(PylRS), ACPK-RS, from M. barkeri shows an improved performance for the expression of recombinant protein backbone oxoesters. By using HdeA as a model protein we demonstrate that the hydrazide-based method can be used to synthesize proteins with correctly folded structures and full biological activity. Because the PylRS-tRNACUAPyl system is compatible with both prokaryotic and eukaryotic cells,the strategy presented here may be readily expanded to manipulate proteins produced in mammalian cells. The new hydrazide-based method may also supplement the intein-based expressed protein ligation method by allowing for a more flexible selection of ligation site.
authors.affiliation: Tsinghua-Peking Center for Life Sciences, Department of Chemistry, Tsinghua University, Beijing 100084, China.
authors.forename: Yi-Ming
authors.initials: YM
authors.lastname: Li
authors.forename: Mai-Yun
authors.initials: MY
authors.lastname: Yang
authors.forename: Yi-Chao
authors.initials: YC
authors.lastname: Huang
authors.forename: Yi-Tong
authors.initials: YT
authors.lastname: Li
authors.forename: Peng R
authors.initials: PR
authors.lastname: Chen
authors.forename: Lei
authors.initials: L
authors.lastname: Liu
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006834:Hydrazines / Q000737:chemistry* / Q000378:metabolism; D006880:Hydroxy Acids / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D006834:Hydrazines; D006880:Hydroxy Acids; D011994:Recombinant Proteins; C084501:hdeA protein, E coli