title: Artificial extracellular matrix proteins containing phenylalanine analogues biosynthesized in bacteria using T7 expression system and the PEGylation.
abstract: In vivo incorporation of phenylalanine (Phe) analogues into an artificial extracellular matrix protein (aECM-CS5-ELF) was accomplished using a bacterial expression host that harbors the mutant phenylalanyl-tRNA synthetase (PheRS) with an enlarged binding pocket. Although the Ala294Gly/Thr251Gly mutant PheRS (PheRS**) under the control of T5 promoter allows incorporation of some Phe analogues into a protein, the T5 system is not suitable for material science studies because the amount of materials produced is not sufficient due to the moderate strength of the T5 promoter. This limitation can be overcome by using a pair of T7 promoter and T7 RNA polymerase instead. In the T7 expression system, it is difficult, however, to achieve a high incorporation level of Phe analogues, due to competition of Phe analogues for incorporation with the residual Phe that is required for synthesis of active T7 RNA polymerase. In this study, we prepared the PheRS** under T7 promoter and optimized culture condition to improve both the incorporation level of recombinant aECM protein and the incorporation level of Phe analogues. Incorporation and expression levels tend to increase in the case of p-azidophenylalanine, p-iodophenylalanine, and p-acetylphenylalanine. We evaluated the lower critical transition temperature, which is dependent on the incorporation ratio and the turbidity decreased when the incorporation level increased. Circular dichromism measurement indicated that this tendency is based on conformational change from random coil to β-turn structure. We demonstrated that polyethylene glycol (PEG) can be conjugated at reaction site of Phe analogues incorporated. We also demonstrated that the increased hydrophilicity of elastin-like sequences in the aECM-CS5-ELF made by PEG conjugation could suppress nonspecific adhesion of human umbilical vein endothelial cells (HUVEC).
authors.affiliation: Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan. takasu.akinori@nitech.ac.jp
authors.forename: Akinori
authors.initials: A
authors.lastname: Takasu
authors.forename: Shiori
authors.initials: S
authors.lastname: Kondo
authors.forename: Akihiro
authors.initials: A
authors.lastname: Ito
authors.forename: Yuya
authors.initials: Y
authors.lastname: Furukawa
authors.forename: Masahiro
authors.initials: M
authors.lastname: Higuchi
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Kinoshita
authors.forename: Inchan
authors.initials: I
authors.lastname: Kwon
mesh_terms: D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects; D006801:Humans; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D010652:Phenylalanine-tRNA Ligase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D010957:Plasmids; D011092:Polyethylene Glycols / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D012360:RNA, Transfer, Phe / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D023822:Tissue Engineering / Q000379:methods*; D014169:Transformation, Bacterial; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D016326:Extracellular Matrix Proteins; D012360:RNA, Transfer, Phe; D011994:Recombinant Proteins; D014764:Viral Proteins; D011092:Polyethylene Glycols; D010649:Phenylalanine; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D010652:Phenylalanine-tRNA Ligase