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Protein–Nucleic Acid Conjugation with Sterol Linkers Using Hedgehog Autoprocessing
journal contribution
posted on 2019-10-10, 19:43 authored by Xiaoyu Zhang, Zihan Xu, Dina S. Moumin, Daniel A. Ciulla, Timothy S. Owen, Rebecca A. Mancusi, José-Luis Giner, Chunyu Wang, Brian P. CallahanHedgehog
(Hh) precursor proteins contain an autoprocessing domain
called HhC whose native function is protein cleavage and C-terminal
glycine sterylation. The transformation catalyzed by HhC occurs in cis from a precursor protein and exhibits wide tolerance
toward both sterol and protein substrates. Here, we repurpose HhC
as a 1:1 protein–nucleic acid ligase, with the sterol serving
as a molecular linker. A procedure is described for preparing HhC-active
sterylated DNA, called steramers, using aqueous compatible chemistry
and commercial reagents. Steramers have KM values of 7–11 μM and reaction t1/2 values of ∼10 min. Modularity of the HhC/steramer
method is demonstrated using four different proteins along with structured
and unstructured sterylated nucleic acids. The resulting protein–DNA
conjugates retain the native solution properties and biochemical function.
Unlike self-tagging domains, HhC does not remain fused to the conjugate;
rather, enzymatic activity is mechanistically coupled to conjugate
release. That unique feature of HhC, coupled with efficient kinetics
and substrate tolerance, may ease access and open new applications
for these suprabiological chimeras.
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repurpose HhCprecursor proteinautoprocessing domainsubstrate toleranceprecursor proteinsprotein substratesHhC-active sterylated DNAsolution propertiesconjugate releaseC-terminal glycine sterylationHedgehog Autoprocessing HedgehogK M valuessuprabiological chimerasprotein cleavageSterol Linkersself-tagging domains
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