Light-responsive bioconjugates as novel tools for specific capture of biologicals by photoaffinity precipitation.
Light-responsive bioconjugates are synthesized by a two-step protocol calling first for cotelomerization (chain-transfer polymerization) of N-isopropylacrylamide and N-acryloxysuccinimide. The desired bioligand (biotin) is used in modified form as chain-transfer agent in this step. As a consequence, 100% of the produced bioconjugates carry this group. In a second step, the cotelomers (bioconjugates) are rendered photoresponsive by linking a chromophore ((3-aminopropyloxy)azobenzene) group to the N-acryloxysuccinimide side chains. The resulting structures show a critical solution temperature in pure water of 16 degrees C when the azo groups in the side chains are predominately in the (stable) trans state. Irradiation with UV light (330 nm) switches the azo group into the more hydrophilic cis state, and the critical solution temperature rises to 18 degrees C. Irradiation with visible light (> 440 nm) switches the group back to the trans state. Adjusting the temperature to an intermediate level, the bioconjugates are used to demonstrate the concept of photo affinity precipitation, i.e., the specific capture and recovery by light-induced precipitation of a target molecule (avidin) from a serum-containing cell-culture supernatant. The avidin was obtained in highly purified form; no nonspecific copurification of protein impurities was observable.
Desponds
Arnaud
A
Laboratory of Chemical Biotechnology, Faculty of Basic Sciences, Swiss Federal Institute of Technology Lausanne, Switzerland.
Freitag
Ruth
R
D000178:Acrylamides / Q000737:chemistry / Q000528:radiation effects; D000179:Acrylates / Q000737:chemistry / Q000528:radiation effects; D000818:Animals; D001360:Avidin / Q000737:chemistry; D001391:Azo Compounds / Q000737:chemistry / Q000528:radiation effects; D001710:Biotin / Q000737:chemistry; D016466:CHO Cells; D011232:Chemical Precipitation; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry; D008024:Ligands; D008027:Light; D013058:Mass Spectrometry; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D010777:Photochemistry; D011108:Polymers / Q000737:chemistry / Q000528:radiation effects*; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization; D013696:Temperature; D014466:Ultraviolet Rays
D000178:Acrylamides; D000179:Acrylates; D001391:Azo Compounds; D003470:Culture Media; D008024:Ligands; D011108:Polymers; D001360:Avidin; D001710:Biotin; C067295:N-isopropylacrylamide