Soluble Expression and Purification of Q59L Mutant L-asparaginase in the Presence of Chaperones in SHuffle™ T7 strain

Background and Aims: Q59L mutant of L-asparaginase enzyme from Escherichia coli (E. coli) has been introduced with lower side effects. This version of the enzyme might have potential applications in the treatment of leukemia patients. We utilized SHuffle T7 strain of E. coli, to produce the mutant enzyme in the presence of chaperone molecules. Materials and Methods: Q59LAsp gene was cloned into pET28a expression vector, and two strains of E. coli (BL21 DE3 and SHuffle T7 strains) were used to produce recombinant protein. In parallel, PG-Tf2 plasmid was cloned into the same strains, and the effect of trigger factor chaperone and groELS chaperonines was studied. The his-tagged recombinant protein was expressed and purified using nickel affinity chromatography. The amount of recombinant protein which is produced in each condition was determined and compared. Results: The amount of soluble recombinant protein was enhanced in the presence of chaperones in both strains of E. coli. SHuffle T7 strain produced more soluble recombinant protein in the soluble state than BL21 DE3 strain. So the best condition for the production of soluble recombinant Q59L mutant protein was the use of PG-Tf2 plasmid in the SHuffle T7 strain. Conclusion: Application of the new strain SHuffle T7, with chaperones simultaneously showed better results in the production of Q59L mutant version of L-Asparaginase.

Spectrophotometric method for evaluating proteolysis in cheeses and aromatic additives with a cheesy taste

The spectrophotometric method for measuring protein content can be used to evaluate the degree of proteolysis in cheeses. At a wavelength of 280 nm, tryptophan and tyrosine are absorbed, a high amount of them is found in casein, the main protein of cheese mass. It was found that the value of the absorbance coefficient of the solution of proteins extracted from flavoring additives with cheese flavor (FA) and cheeses depends on the degree of proteolysis of proteins in the cheese mass and differs in FA and different types of cheeses. The highest absorbance coefficient is observed in the FA samples A1%1cm = 10.30, in which from 65 to 81% of the protein is converted into a soluble state. In cheeses, the degree of proteolysis is from 23 to 33%, and the absorbance coefficient of solution is from 1.1 to 2.4 (with the exception of Cheddar cheese), which indicates an incomplete transition of amino acids absorbing radiation at 280 nm into the extract released from cheeses. Using the spectrophotometric method, the results of measuring the content of soluble protein in cheeses and FA, strictly correlating with the results achieved by the Kjeldahl method (R2 > 0.81), can be obtained. To get reliable results of evaluating the content of water-soluble protein in cheeses, it is necessary to carry out measurements on a sample of cheeses belonging to the same species group, having the same specificity of proteolysis and slightly different absorbance coefficient between samples within the instance.

Arsenic in Pteris vittata is localized to the cell wall in a water-soluble state

Pteris vittata L., a hyperaccumulator of As, has great potential in environmental remediation. Localization analysis of As and Ca, K, S, and P in arsenic-exposed and unexposed P. vittata pinnae was performed using an electron probe micro analyzer. A comparison of section preparation methods revealed that the As in P. vittata is water-soluble and not tightly bound to the tissue. Further elemental mapping revealed that the localization of S was not consistent with that of As. Therefore, the formation of a complex of low-molecular-weight thiols and As, which is a common detoxification mechanism in plants, may be a limited in P. vittata. It was confirmed that As was localized around the cell wall when the cell was in a healthy state, and that the distribution of As expanded to the whole cell when the cell was damaged. These results suggest that P. vittata may retain As extracellularly to inhibit damage to healthy cells. However, in the periphery of pinnae, which is more susceptible to damage, As was distributed throughout the cell, eventually leading to browning.

DNAJB12 and Hsp70 Triage Arrested Intermediates of N1303K-CFTR for ER Associated-Autophagy

The transmembrane Hsp40 DNAJB12 and cytosolic Hsp70 cooperate on the ER's cytoplasmic face to facilitate the triage of nascent polytopic membrane proteins for folding versus degradation. N1303K is a common mutation that causes misfolding of the ion channel CFTR, but unlike F508del-CFTR, biogenic and functional defects in N1303K-CFTR are resistant to correction by folding modulators. N1303K is reported to arrest CFTR folding at a late stage after partial assembly of its N-terminal domains. N1303K-CFTR intermediates are clients of JB12-Hsp70 complexes, maintained in a detergent soluble-state, and have a relatively long 3-hour half-life. ERAD-resistant pools of N1303K-CFTR are concentrated in ER-tubules that associate with autophagy initiation sites containing WIPI1, FlP200, and LC3. Destabilization of N1303K-CFTR or depletion of JB12 prevents entry of N1303K-CFTR into the membranes of ER-connected phagophores and traffic to autolysosomes. In contrast, the stabilization of intermediates with the modulator VX-809 promotes the association of N1303K-CFTR with autophagy initiation machinery. N1303K-CFTR is excluded from the ER-exit sites, and its passage from the ER to autolysosomes does not require ER-phagy receptors. DNAJB12 operates in biosynthetically active ER-microdomains to triage membrane protein intermediates in a conformation-specific manner for secretion versus degradation via ERAD or selective-ER-associated autophagy.

Small-molecule sequestration of amyloid-β as a drug discovery strategy for Alzheimer’s disease

Disordered proteins are challenging therapeutic targets, and no drug is currently in clinical use that modifies the properties of their monomeric states. Here, we identify a small molecule (10074-G5) capable of binding and sequestering the intrinsically disordered amyloid-β (Aβ) peptide in its monomeric, soluble state. Our analysis reveals that this compound interacts with Aβ and inhibits both the primary and secondary nucleation pathways in its aggregation process. We characterize this interaction using biophysical experiments and integrative structural ensemble determination methods. We observe that this molecule increases the conformational entropy of monomeric Aβ while decreasing its hydrophobic surface area. We also show that it rescues a Caenorhabditis elegans model of Aβ-associated toxicity, consistent with the mechanism of action identified from the in silico and in vitro studies. These results illustrate the strategy of stabilizing the monomeric states of disordered proteins with small molecules to alter their behavior for therapeutic purposes.

DNAJB12 and Hsp70 Facilitate the Conformation Specific Degradation of Arrested N1303K-CFTR Intermediates by ER Associated-Autophagy

SUMMARYThe transmembrane Hsp40 DNAJB12 and cytosolic Hsp70 cooperate on the ER’s cytoplasmic face to facilitate the triage of nascent polytopic membrane proteins for folding versus degradation. N1303K is the second most common mutation in the ion channel CFTR, but unlike F508del-CFTR, biogenic and functional defects in N1303K-CFTR are resistant to correction bolding modulators. N1303K is reported to arrest CFTR folding at a late stage after partial assembly of its N-terminal domains. N1303K-CFTR intermediates are clients of JB12-Hsp70 complexes, maintained in a detergent soluble-state, and have a relatively long 3-hour half-life. ERAD-resistant pools of N1303K-CFTR are concentrated in ER-tubules that associate with autophagy initiation sites containing WIPI1, FlP200, and LC3. Destabilization of N1303K-CFTR or depletion of JB12 prevents entry of N1303K-CFTR into the membranes of ER-connected phagophores and autolysosomes. Whereas, the stabilization of intermediates with the modulator VX-809 promotes the association of N1303K-CFTR with autophagy initiation machinery. N1303K-CFTR is excluded from the ER-exits site, and its passage from the ER to autolysosomes does not require ER-phagy receptors. DNAJB12 operates in biosynthetically active ER-microdomains to triage in a conformation-specific manner membrane protein intermediates for secretion versus degradation via ERAD or selective-ER associated autophagy.

RETURNABLE BAKING WASTE — A NEW TYPE OF RAW MATERIALS FOR DISTILLATES PRODUCTION (PART II. STAGE OF RAW MATERIALS PREPARATION FOR DISTILLATION)

The processes that take place upon saccharified wort obtaining from the returnable baking waste and its fermentation was the research subject of this work. The development of operational parameters at the stage of returnable baking waste preparation for distillation, which provides a high-quality product is the purpose of the work. The samples of saccharified and fermented wort obtained from various bread and bakery products types produced by large enterprises in Moscow were the objects of the study. To characterize the composition of saccharified and fermented wort, the indicators to assess the quality of the wort from grain raw materials were used. The mass concentration of individual sugars in the wort was determined using high performance liquid chromatography on an Agilent Technologies 1200 Series device. The qualitative composition and volatile components concentration in the fermented wort was determined using gas chromatography on a Thermo Trace GC Ultra device. It was established that the percentage of solids transition to a soluble state does not depend on a returnable waste type and is in the range from 87.6% to 90.7%, and the starch transition to a soluble state, on the contrary, is determined by the processed raw materials type. It is shown that the use of rye-wheat bread after its preliminary enzymatic treatment with thinning and cytolytic drugs in a mixture with wheat bread in a ratio of 1÷1 to 1÷2 can improve the wort rheological characteristics and transfer from 98.1% to 99.3% starch of raw materials in a soluble state. It has been shown that for the efficient process of saccharified wort fermentation from returnable baking waste, the Fermiol and Turbo‑24 alcohol yeast races are most suitable, which ensure a high yield of ethanol and an optimal composition of volatile components in the fermented wort.

Development of the Method to Produce Functionally Active Recombinant Streptavidin in Escherichia coli Cells

Streptavidin is a homotetrameric protein produced by Streptomyces avidinii, each subunit of which binds biotin (vitamin H), forming a stable complex (Kd = 10-15 М). Streptavidin-biotin coreaction is widely used in analytical systems, for targeted delivery of compounds, for affinity purification, etc. The aim of this study was to develop a rational technique to produce functionally active recombinant streptavidin. Recombinant Escherichia coli strains producing minimal core and full-sized streptavidin variants were obtained. The E. coli BL21 Codon Plus (DE3) RIPL, as host cells, and the pET19b plasmid carrying gene of minimally-sized core (miniSAV) or full-sized (SAV) streptavidin were used. Synthesis of miniSAV results in its localization as insoluble inclusion bodies. Denatured miniSAV yield was 130 mg per liter of E. coli c ulture. T he r enaturation g ives o nly 10- 15 % of the functionally active protein. Full-sized streptavidin localizes in the cytoplasm in a soluble state, but its toxicity causes low yield of the protein (10-13 mg per liter of the culture). The induction of SAV synthesis at the end of the logarithmic stage of cell growth was found to increase the yield of SAV approximately 2-fold. The yield of functionally active protein was 30 mg per liter culture. SAV was produced practically in individual state after affine chromatography on 2-iminobiotin agarose. One molecule of full-sized streptavidin bound 3.9 biotin molecules as was shown by colorimetric analysis using HABA (4-hydroxyazobenzene-2-carboxylic acid). Both streptavidins form sandwichtype complexes with biotinylated molecules in solid-phase microassay conditions. E. coli BL21 Codon Plus (DE3) RIPL/pET19bSAV strain was stable during storage with 20 % glycerol at -70 °С, which was shown by repeated two-year reseeding. The streptavidin producing strain (E. coli BL21 Codon Plus (DE3) RIPL/pET19bSAV) is deposited in the Collection for extremophile microorganisms and type cultures (Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk), No. 3505. The method for producing functionally active recombinant streptavidin developed in this study ensures its availability for biotechnological research