We
report a simple temperature-responsive bioconjugate system comprising
superfolder green fluorescent protein (sfGFP) decorated with poly[(oligo
ethylene glycol) methyl ether methacrylate] (PEGMA) polymers. We used
amber suppression to site-specifically incorporate the non-canonical
azide-functional amino acid p-azidophenylalanine
(pAzF) into sfGFP at different positions. The azide
moiety on modified sfGFP was then coupled using copper-catalyzed “click”
chemistry with the alkyne terminus of a PEGMA synthesized by reversible
addition–fragmentation chain transfer (RAFT) polymerization.
The protein in the resulting bioconjugate was found to remain functionally
active (i.e., fluorescent) after conjugation. Turbidity measurements
revealed that the point of attachment of the polymer onto the protein
scaffold has an impact on the thermoresponsive behavior of the resultant
bioconjugate. Furthermore, small-angle X-ray scattering analysis showed
the wrapping of the polymer around the protein in a temperature-dependent
fashion. Our work demonstrates that standard genetic manipulation
combined with an expanded genetic code provides an easy way to construct
functional hybrid biomaterials where the location of the conjugation
site on the protein plays an important role in determining material
properties. We anticipate that our approach could be generalized for
the synthesis of complex functional materials with precisely defined
domain orientation, connectivity, and composition.
Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes. The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.
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