Protein Modification Through in vivo Incorporation of Noncanonical Amino Acids

noncanonical amino acids;protein engineering

Mock, Marissa Lee ; Tirrell, David A.

University:California Institute of Technology

Department:Chemistry and Chemical Engineering

关键词: noncanonical amino acids;    protein engineering;   

Others  :  https://thesis.library.caltech.edu/4251/34/Mock-M-2006.pdf

美国|英语

来源: Caltech THESIS

【 摘 要 】

Traditional techniques of polymer synthesis produce macromolecules with statistical distributions of chain length, composition, stereochemistry, and sequence.Nature has evolved a complex system for polypeptide synthesis that gives essentially complete control of chain length and monomer sequence.Using the natural protein biosynthesis machinery to produce protein polymers provides not only a unique opportunity to study the effects of such molecular characteristics on material properties, but also the possibility of readily incorporating bioactive domains into protein-based materials.

The objective of this thesis work was to expand upon the set of amino acids available for incorporation into proteins in vivo and to explore applications of the novel chemistries and physical properties provided by the new analogs.

Chapter 2 describes the incorporation of new unsaturated analogues of isoleucine, the alkene 2-amino-3-methyl-4-pentenoic acid and the alkyne 2-amino-3-methyl-4-pentynoic acid, by the wild type E. coli biosynthetic apparatus.Incorporation was found to be sensitive to side chain stereochemistry in the case of the alkene analog; the translational activity of the pairs of enantiomers (SS, RR and SR, RS) were markedly different.We concluded that, although the SS-isomer is a good analogue, the SR-isomer is not incorporated into proteins by this expression host.

Chapter 3 focuses on the incorporation of a fluorine-containing noncanonical amino acid, 5,5,5-trifluoroisoleucine, into artificial extracellular matrix proteins.The fluorinated proteins displayed altered solubility phase behavior and were more resistant to degradation by the physiologically relevant protease elastase, yet retained the ability to adhere endothelial cells in a sequence specific manner.

Chapter 4 describes the incorporation of the photoreactive noncanonical analog p-azidophenylalanine into artificial extracellular matrix proteins.Films of the azide-containing proteins were crosslinked upon short exposure to ultraviolet radiation.Using simple patterned masks, we demonstrated the ability to pattern protein films by only exposing certain regions.When protein patters were produced on a non-adhesive background, endothelial cells selectively adhered to the protein regions to create stable cell patterns.