Īccording to the current reports, MARs may cohabit with core origin replication (ORIs) and another fraction might cohabit with transcriptional enhancers. MARs appear to be functionally conserved, since animal MARs can bind to plant nuclear scaffolds and vice versa. Of the MAR elements reported, many do not display extensive sequence homology, their DNA sequence is highly polymorphic. A set of 21 characteristics is deduced or proposed for MAR/ORI sequences including their enrichment in inverted repeats, AT tracts, DNA unwinding elements, replication initiator protein sites, homooligonucleotide repeats (i.e., AAA, TTT, CCC), curved DNA, DNase I-hypersensitive sites, nucleosome-free stretches, polypurine stretches, and motifs with a potential for left-handed and triplex structures. MARs are about 200 bp long, AT-rich, contain topoisomerase II consensus sequences and other AT-rich sequence motifs, often reside near cis-acting regulatory sequences, and their binding sites are abundant (greater than 10,000 per mammalian nucleus). DNA sequences that bind preferentially to nuclear matrices are named matrix attachment region (MAR) or scaffold associated region (SAR), which were supposed to mediate this loop formation in vivo. Chromatin is looped into domains by attachment of the chromatin fibre to the nuclear matrix.
#Looped domains with protein scaffold free#
This construct self-assembled and when added to T cell culture could stimulate T cell growth to the same degree as free CD28 antibody, indicating its function.Nuclear matrices or nuclear scaffold were defined the biochemical fraction of the nucleus after treatment by with detergent, salt and nucleases, mainly consist of non-histone protein, RNA and DNA in the eukaryote. The authors engineered a 6-loop cTRP construct with a single-chain Fv domain capable of binding CD28. T cell stimulation and growth in vitro requires co-stimulation from its TCR and co-receptors like CD28. This tetrameric cTRP-MHC complex stained CMV-specific T cells to a similar degree to traditional tetramer reagents. The authors used their 6-loop construct to produce cTRP tetramers conjugated to four peptide-MHC complexes presenting a peptide derived from cytomegalovirus (CMV). MHC tetramers are useful immunologic reagents used to identify and stain T cells specific for a certain peptide-MHC complex. Constructs containing peptide-binding or protein ligation domains were able to successfully bind their respective partners, allowing flexibility in loading many cargo types onto cTRP24. Numerous protein cargo types were tested: peptide-binding domains, protein ligation domains, fluorescent proteins, as well as single-chain MHC molecules and single-chain Fv or receptor domains targeting important immune modulators. Next, the authors wanted to test if cargo could be bound to cTRP24 and retain function. Additionally, the authors used a mammalian expression system to broaden their ability to test modifications to cTRP24 that might not be well expressed in bacterial systems. Both the 12-loop and 6-loop constructs were stabilized by this modification and could self-assemble into dimers or tetramers, respectively. The authors sought to stabilize the interaction by modifying the protein termini to contain cysteines allowing disulfide bonds between multimers.
#Looped domains with protein scaffold full#
Only the 12-loop construct could assemble into a dimer of the full 24 looped structure, however dimerization was not complete. The authors modified their construct to express repeats of 3, 4, 6, or 12 loops to see if these smaller units could self-assemble into the full cTRP of 24 repeats. Shaped like a donut, cTRP24 has an outer diameter of approximately 100 angstroms and inner diameter of approximately 60 angstroms. The authors computationally designed and expressed a cTRP with 24 tandem loop repeats, named cTRP24. The results of their engineering efforts were recently published in Nature Structural & Molecular Biology. The Bradley, Stoddard, Riddell and Kaiser labs teamed up to further develop cTRPs with functional domains. Circular TRPs (cTRPs) can be made from repeating motifs, allowing protein cargo to be attached at regular intervals around the structure. Additionally, TRPs can be conjugated to various forms of functional cargo at defined positions. TRPs are useful for protein engineering as they are compact, have high thermal stability, good solubility characteristics, and are relatively easy to design and express. Tandem repeat proteins (TRPs) are small proteins with repeated sequences. Protein engineering offers unique solutions for protein scaffolding for a variety of uses.
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