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Artificial Extracellular Matrix Proteins Containing Phenylalanine Analogues Biosynthesized in Bacteria Using T7 Expression System and the PEGylation

Takasu, Akinori and Kondo, Shiori and Ito, Akihiro and Furukawa, Yuya and Higuchi, Masahiro and Kinoshita, Takatoshi and Kwon, Inchan (2011) Artificial Extracellular Matrix Proteins Containing Phenylalanine Analogues Biosynthesized in Bacteria Using T7 Expression System and the PEGylation. Biomacromolecules, 12 (10). pp. 3444-3452. ISSN 1525-7797. https://resolver.caltech.edu/CaltechAUTHORS:20111107-115431592

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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).


Item Type:Article
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http://dx.doi.org/10.1021/bm200522dDOIArticle
http://pubs.acs.org/doi/abs/10.1021/bm200522dPublisherArticle
Additional Information:© 2011 American Chemical Society. Received: April 18, 2011; revised: July 29, 2011; published: August 8, 2011. A.T. is grateful to Prof. David A. Tirrell for the opportunity to work in his laboratory at California Institute of Technology. The authors acknowledge support from the Nagoya Institute of Technology’s Research Promotion Program, from NIH Grants EB1971 and GM 62523, and from the NSF Center for the Science and Engineering of Materials at the California Institute of Technology. We thank Dr. Hideo Yoshizato and Mr. Yuya Furukawa for technical assistance.
Funders:
Funding AgencyGrant Number
Nagoya Institute of TechnologyUNSPECIFIED
NIHEB1971
NIHGM 62523
NSFUNSPECIFIED
Issue or Number:10
Record Number:CaltechAUTHORS:20111107-115431592
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20111107-115431592
Official Citation:Artificial Extracellular Matrix Proteins Containing Phenylalanine Analogues Biosynthesized in Bacteria Using T7 Expression System and the PEGylation Akinori Takasu, Shiori Kondo, Akihiro Ito, Yuya Furukawa, Masahiro Higuchi, Takatoshi Kinoshita, and Inchan Kwon Biomacromolecules 2011 12 (10), 3444-3452
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:27652
Collection:CaltechAUTHORS
Deposited By: Jason Perez
Deposited On:08 Nov 2011 20:44
Last Modified:03 Oct 2019 03:25

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