7 research outputs found
Amberlite XAD-4 is a convenient tool for removing Triton X-100 and Sarkosyl from protein solutions
No full textAmberlite has been shown to be an appropriate material for the adsorption of organic contaminants from aqueous solutions. In addition, Amberlite XAD-2 has been successfully used, as an alternative to Bio-Beads, to remove Triton X-100 from protein solutions, such as from samples of solubilized membrane proteins. However, Amberlite has not been tested as an adsorbent when a mixture of detergents is necessary to solubilize and refold a target protein. Here the authors show that Amberlite XAD-4 can be appropriately used to aid the purification process of proteins solubilized from inclusion bodies with the ternary detergent system consisting of Sarkosyl, Triton X-100 and CHAPS
High-yield production in Escherichia coli and convenient purification of a candidate vaccine against SARS-CoV-2
No full textObjectives: The aim of the present work was to identify a time-saving, effective, and low-cost strategy to produce in Escherichia coli a protein chimera representing a fusion anti-SARS-CoV-2 candidate vaccine, consisting of immunogenic and antigenic moieties.
Results: We overexpressed in E. coli BL21(DE3) a synthetic gene coding for CRM197-RBD, and the target protein was detected in inclusion bodies. CRM197-RBD was solubilized with 1 % (w/v) of the anionic detergent N-lauroylsarcosine (sarkosyl), the removal of which from the protein solution was conveniently accomplished with Amberlite XAD-4. The detergent-free CRM197-RBD was then separated from contaminating DNA using polyethylenimine (PEI), and finally purified from PEI by salting out with ammonium sulfate. Structural (CD spectrum) and functional (DNase activity) assays revealed that the CRM197-RBD chimera featured a native and active conformation. Remarkably, we determined a yield of purified CRM197-RBD equal to 23 mg per litre of culture.
Conclusions: To produce CRM197-RBD, we devised the use of sarkosyl as an alternative to urea to solubilize the target protein from E. coli inclusion bodies, and the easy removal of sarkosyl by means of Amberlite XAD-4
(Ne)poznavanje slovenstva v južni Italiji
No full textČeprav smo Slovenci po mnenju angleškega zgodovinarja A. J. P. Taylorja t. i. "kulturni narod", nas v južni Italiji kaj malo poznajo, saj so prevodi, ki sežejo do tja, zlasti iz beletristike. Avtorica vidi (ne)poznavanje slovenstva po letu 2000 v prevodni književnosti, kjer je bogata gledališka dejavnost na jugu popolnoma odsotna.Even though Slovenia, according to the English historian A. J. P. Taylor, is a "cultural nation", Slovene literature is not very well known in Southern Italy. Only translations of some major works of fiction can be found. Furthermore, the author observes the inadequacy of translated literature after 2000, with the rich theatrical activity in the south being entirely absent
Minimal shuttle vectors for <i>Saccharomyces cerevisiae</i>
No full textSophisticated genetic engineering tasks such as protein domain grafting and multi-gene fusions are hampered by the lack of suitable vector backbones. In particular, many restriction sites are in the backbone outside the polylinker region (multiple cloning site; MCS) and thus unavailable for use, and the overall length of a plasmid correlates with poorer ligation efficiency. To address this need, we describe the design and validation of a collection of six minimal integrating or centromericshuttle vectors for Saccharomyces cerevisiae, a widely used model organism in synthetic biology. We constructed the plasmids using de novogene synthesis and consisting only of a yeast selection marker (HIS3, LEU2, TRP1, URA3, KanMX, or natMX6), a bacterial selection marker (Ampicillin resistance), an origin of replication (ORI), and the MCS flanked by M13 forward and reverse sequences. We use truncated variants of these elements where available and eliminated all other sequences typically found in plasmids. The MCS consists of ten unique restriction sites. To our knowledge, at sizes ranging from approximately 2.6 kb to 3.5 kb, these are the smallest shuttle vectors described for yeast. Further, we removed common restriction sites in the open reading frames (ORFs) and terminators, freeing up approximately 30 cut sites in each plasmid. We named our pLS series in accordance with the well-known pRS vectors, which are on average 63% larger: pLS400, pLS410(KanMX); pLS403, pLS413(HIS3); pLS404, pLS414(TRP1); pLS405, pLS415(LEU2); pLS406, pLS416(URA3); pLS408, pLS418(natMX6). This resource substantially simplifies advanced synthetic biology engineering in Saccharomyces cerevisiae.UPBARTHLPB
Supplementary Materials: Amberlite XAD-4 is a convenient tool for removing Triton X-100 and Sarkosyl from protein solutions.pdf
No full textSupplementary Materials</p
De novo design of light-regulated dynamic proteins using deep learning
No full textRecent advances in deep learning have enabled accurate design of static protein structures, but the de novo design of protein functions controlled by programmable, intramolecular conformational changes remains an unsolved challenge. Here, we present a general deep learning-guided framework for designing dynamic, multi-domain proteins allosterically regulated by light. By integrating photoresponsive domains into de novo scaffolds, we engineered conformational switches that exhibit precise, reversible structural transitions upon illumination. Structural, spectroscopic, and functional analyses validated our designs and demonstrated precise spatio-temporal optogenetic control of diverse cellular processes, including subcellular localization, intercellular signaling, and population-level behaviors. This work establishes a broadly applicable strategy for encoding long-range allosteric control through designed intramolecular motions, and opens new avenues for programming dynamic protein functions from first principles, with implications for basic research, synthetic biology, and therapeutic development.UPBARTHUPDALPEPTPSPLPB
Deep learning-based joint sequence–structure <i>de novo</i> membrane protein design
No full textDeep learning has revolutionized soluble protein design, yet de novo transmembrane (TM) protein engineering remains hindered by scarce structural data, complex membrane-specific interactions and conformational dynamics. We developed TMDiffusion (TMDF), a joint all-heavy-atom sequence–structure diffusion model trained to capture the full interaction diversity of natural TM proteins, including weak and polar contact networks. TMDF designs diverse TM architectures—associating domains, inhibitors, and conformational switches—in a single step, achieving >70% experimental success. A crystal structure of designed proteins matches predictions with atomic accuracy. Leveraging TMDF, we built synthetic single-pass receptors whose de novo TM domains toggle between conformations, enabling precise control of signalling outputs consistent with predicted equilibria. These results show that membrane-adapted DL models can accurately encode and program TM association energetics and conformations. TMDF establishes a general framework for bottom-up design of TM proteins with programmable functions, advancing both mechanistic studies of membrane proteins and development of next-generation therapeutics.UPBART
