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Recombinant Antibodies
Published in Siegfried Matzku, Rolf A. Stahel, Antibodies in Diagnosis and Therapy, 2019
Melvyn Little, Sergey M. Kipriyanov
It is now recognized that aggregation in vivo is not a function of the solubility and stability of the native state of the protein, but of those of its folding intermediates in their particular environment (Hockney, 1994; Plückthun, 1994). However, the overexpression of some enzymes of the E. coli folding machinery such as GroES/L chaperonins, disulfide-isomerase DSbA and proline-cis-trans-isomerase (PPIase) did not increase the yield of soluble antibody fragments (Knappik et al., 1993; Duenas et al., 1994). The degree of successful in vivo folding appears to depend very much on the primary sequence of the variable domains (Plückthun, 1994; Knappik and Plückthun, 1995).
Shigella: Insights into the Clinical Features, Pathogenesis, Diagnosis, and Treatment Strategies
Published in Dongyou Liu, Handbook of Foodborne Diseases, 2018
Periyanaina Kesika, Bhagavathi Sundaram Sivamaruthi, Krishnaswamy Balamurugan
Shigella move rapidly in the epithelial cytosol by means of actin-based motility, which is mediated by the outer membrane protein IcsA.48 SopA is a protease that is required for the secretion and localization of IcsA at one pole of Shigella.49 The actin-based motility and interaction of cadherin with the actin tail provide a driving force for the formation of a rigid protrusion structure at the intermediate junction of epithelial cells.50 After the protrusion entered the neighboring epithelial cell, Shigella rapidly destroy the double membrane–bounded compartment (protrusion) using a similar mechanism involved for the disruption of a single-membrane vacuole in an epithelial cell. Also, the bacterial periplasmic protein disulfide bond catalyst DsbA is required for the oxidative folding of Spa32, which is a component of the T3S system. The thiol sulfide oxidoreductase, DsbA-mediated Spa32 folding is essential for Shigella, which aids in the secretion of Ipa effector proteins and lysis of interepithelial protrusions formed during cell-to-cell spread.51–53 Along with the effectors IpaB and IpaC, cytosolic chaperone, IpgC (invasion plasmid gene) are involved in the lysis of double-membrane vacuole (protrusion membranes).54,55
Polyethyleneimine-polyethylene glycol copolymer targeted by anti-HER2 nanobody for specific delivery of transcriptionally targeted tBid containing construct
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Batoul Saqafi, Fatemeh Rahbarizadeh
A modified version of the pNIC28-DsbA1 vector was used in order to clone DsnA2-RR-4 where the researchers decided to neglect the export signal sequence. The above-said vector is responsible for encoding DsbA (periplasmic protein disulfide isomerase I). Next, you could observe a sequence of an N-terminal hexahistidine tag and a TEV protease recognition site (amino acid sequence ENLYFQ*S) and two unique BsaI sites for ligation-independent cloning (LIC). Utilizing standard ligation-independent cloning (LIC) protocols [20], those DNA sequences which encoded the proteins of interest flanked by LIC sites were PCR amplified, T4 polymerase-treated and cloned into expression vectors. A mismatch was designed in TEV P2’ position so that it could be mutated to a serine. LB agar plates that contained 50 μg/ml kanamycin and 5% sucrose were utilized to select those vectors containing the insert. The sequences of plasmids were verified before they were transformed into expression strain BL21 (DE3)-R3-pRARE2.
YjbH mediates the oxidative stress response and infection by regulating SpxA1 and the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) in Listeria monocytogenes
Published in Gut Microbes, 2021
Changyong Cheng, Xiao Han, Jiali Xu, Jing Sun, Kang Li, Yue Han, Mianmian Chen, Houhui Song
L. monocytogenes thrives in dramatically distinct environments during its transition from saprophyte to cytosolic pathogen.37 The intracellular lifecycle of Listeria requires it to survive the harsh phagosomal compartment, escape into the highly reducing cytosol, and spread to neighboring cells.12 Proteins in the thioredoxin family play major roles in the protection of cells against toxic oxygen species as well as maintaining the bacterial thiol–disulfide balance. Here, we investigated the roles of thioredoxin-like YjbH in the adaptation of L. monocytogenes to diverse redox environments and, more importantly, in the regulation of PTS and virulence during infection. Based mostly on studies in Bacillus, YjbH is known as an adaptor protein that targets Spx for ClpXP protease-mediated degradation. Here, for the first time, we demonstrated that YjbH contributes to the oxidative stress response and is able to interact with SpxA1, the Spx family member that is required for the oxidative stress response and pathogenesis of L. monocytogenes.5 Interestingly, L. monocytogenes YjbH was found required for the metal ion-induced oxidative stress response but not for the response to stress induced by H2O2 or the thiol-specific oxidant, diamide. Cu2+ and Cd2+ sensitivity assays are usually performed to assess oxidase and isomerase activities, respectively.38 As we previously determined that L. monocytogenes encodes a complete thioredoxin system (TrxA and TrxB) that participates in response to diamide-induced oxidative stress,22 we speculate that, unlike other bacterial species, L. monocytogenes has a delicate division of responsibilities in defending against different kinds of oxidative stress and YjbH might act as a disulfide bond formation protein (DSB) that has both disulfide oxidase and isomerase activities. Many Gram-positive bacteria have different complements of DSB proteins. Searches for orthologs of B. subtilis DsbA and DsbB (known as BdbD and BdbC, respectively) in low-G + C Gram-positive bacteria revealed that many of these organisms contain either a DsbA or DsbB protein, but not both.39,40L. monocytogenes encodes two putative DSB proteins, Lmo0964 (i.e., YjbH) and Lmo1059, annotated as DsbA-like and DsbG, respectively, in the GenBank database. However, Lmo0964 was recently designated as a putative thioredoxin similar to B. subtilis YjbH and shown to contribute to expression of the ActA protein, which is required for L. monocytogenes actin-based motility.11 Based on this, together with our findings, we suggest that YjbH functions as an adaptor by interacting with Spx and also as a DSB protein, which has an essential role in the oxidative stress response and correct oxidative stress-induced protein folding in L. monocytogenes (Figure 5).