| 1. |
- Elfageih, Rageia, et al.
(författare)
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Characterization of the Streptococcus pneumoniae neuraminidases NanA, NanB, and NanC
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Streptococcus pneumoniae is a Gram-positive bacterium that is commonly found in the upper respiratory tract of humans where it obtains nutrients for growth from the mucosal environment. The genome of S. pneumoniae encodes up to three different neuraminidases, i.e., NanA, NanB and NanC. Neuraminidases or sialidases enable pathogens to cleave the terminal sialic acid from carbohydrate complexes in the mucous and on the host cell surface. Sialic acid removal via neuraminidases often exposes galactose residues and other underlying sugars that together with sialic acid can be metabolized by the bacterium. Here, we have started a comparative characterization of NanA, NanB, and NanC. The three proteins were produced in the cytoplasm of E. coli as GST fusion proteins. The fusion proteins were isolated and the mature full-length NanA, NanB, and NanC were separated from the GST by proteolysis using the tobacco etch virus (TEV) protease. We characterized the oligomeric state of the recovered NanA, NanB, and NanC proteins using size exclusion chromatography and blue native PAGE and also determined the pH optimum for the catalytic activity using 2′-(4-methylumbelliferyl)-α-D-N- acetylneuraminic acid (MUNANA) as a substrate. Our data indicate that NanA, NanB, and NanC primarily form monomers and that the catalytic activity of NanA and NanB is most active at a pH of 6.6, while NanC catalysis appears to favor a pH of 7
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| 2. |
- Elfageih, Rageia, et al.
(författare)
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Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli
- 2020
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 29:10, s. 2028-2037
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Tidskriftsartikel (refereegranskat)abstract
- Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond-containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild-type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding-induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of similar to 160 angstrom, and that PhoA appears to fold cotranslationally via at least two disulfide-stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra-cytosolic compartment, like the periplasm.
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| 3. |
- Elfageih, Rageia, 1980-
(författare)
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Production and folding of proteins in the periplasm of Escherichia coli
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Gram-negative bacterium E. coli is the most widely used host for the production of recombinant proteins. Disulfide bond containing recombinant proteins are usually produced in the periplasm of E. coli since in this compartment of the cell - in contrast to the cytoplasm - disulfide bond formation is promoted. To reach the periplasm recombinant proteins have to be translocated across the cytoplasmic membrane by the protein translocation machinery. To obtain sufficient yields of active recombinant protein in the periplasm is always challenging. The Ph.D. studies have aimed at developing strategies to enhance recombinant protein production yields in the periplasm, to better understand what happens when a protein is produced in the periplasm, and to shed light on the protein folding process in the periplasm. It has been shown that evolving translation initiation regions (TIRs) can enhance periplasmic protein production yields of a variety of proteins. Furthermore, it has been shown that the protein translocation machinery can adapt for enhanced periplasmic recombinant protein production. Force profile analysis was used to study co-translational folding of the periplasmic disulfide-bond containing protein alkaline phosphatase (PhoA) in the periplasm. It was shown that folding-induced forces can be transmitted via the nascent chain from the periplasm to the peptidyl transferase center in the ribosome and that PhoA appears to fold co- translationally via disulfide-stabilized folding intermediates. Finally, the S. pneumoniae neuraminidases NanA, NanB, and NanC were produced in E. coli and subsequently isolated. The activity of these neuraminidases was monitored at different pH as well as their oligomeric state was studied.
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| 4. |
- Karyolaimos, Alexandros, et al.
(författare)
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Escherichia coli Can Adapt Its Protein Translocation Machinery for Enhanced Periplasmic Recombinant Protein Production
- 2020
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Ingår i: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media SA. - 2296-4185. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Recently, we engineered a tunable rhamnose promoter-based setup for the production of recombinant proteins in E. coli. This setup enabled us to show that being able to precisely set the production rate of a secretory recombinant protein is critical to enhance protein production yields in the periplasm. It is assumed that precisely setting the production rate of a secretory recombinant protein is required to harmonize its production rate with the protein translocation capacity of the cell. Here, using proteome analysis we show that enhancing periplasmic production of human Growth Hormone (hGH) using the tunable rhamnose promoter-based setup is accompanied by increased accumulation levels of at least three key players in protein translocation; the peripheral motor of the Sec-translocon (SecA), leader peptidase (LepB), and the cytoplasmic membrane protein integrase/chaperone (YidC). Thus, enhancing periplasmic hGH production leads to increased Sec-translocon capacity, increased capacity to cleave signal peptides from secretory proteins and an increased capacity of an alternative membrane protein biogenesis pathway, which frees up Sec-translocon capacity for protein secretion. When cells with enhanced periplasmic hGH production yields were harvested and subsequently cultured in the absence of inducer, SecA, LepB, and YidC levels went down again. This indicates that when using the tunable rhamnose-promoter system to enhance the production of a protein in the periplasm, E. coli can adapt its protein translocation machinery for enhanced recombinant protein production in the periplasm.
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| 5. |
- Klenow, Laura, et al.
(författare)
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Influenza virus and pneumococcal neuraminidases enhance catalysis by similar yet distinct sialic acid-binding strategies
- 2023
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 299:2
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Tidskriftsartikel (refereegranskat)abstract
- Influenza A viruses and the bacterium Streptococcus pneumoniae (pneumococci) both express neuraminidases that catalyze release of sialic acid residues from oligosaccharides and glycoproteins. Although these respiratory pathogen neuraminidases function in a similar environment, it remains unclear if these enzymes use similar mechanisms for sialic acid cleavage. Here, we compared the enzymatic properties of neuraminidases from two influenza A subtypes (N1 and N2) and the pneumococcal strain TIGR4 (NanA, NanB, and NanC). Insect cell-produced N1 and N2 tetramers exhibited calcium-dependent activities and stabilities that varied with pH. In contrast, E. coli-produced NanA, NanB, and NanC were isolated as calcium insensitive monomers with stabilities that were more resistant to pH changes. Using a synthetic substrate (MUNANA), all neuraminidases showed similar pH optimums (pH 6–7) that were primarily defined by changes in catalytic rate rather than substrate binding affinity. Upon using a multivalent substrate (fetuin sialoglycans), much higher specific activities were observed for pneumococcal neuraminidases that contain an additional lectin domain. In virions, N1 and especially N2 also showed enhanced specific activity toward fetuin that was lost upon the addition of detergent, indicating the sialic acid–binding capacity of neighboring hemagglutinin molecules likely contributes to catalysis of natural multivalent substrates. These results demonstrate that influenza and pneumococcal neuraminidases have evolved similar yet distinct strategies to optimize their catalytic activity.
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| 6. |
- Mirzadeh, Kiavash, et al.
(författare)
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Increased production of periplasmic proteins in Escherichia coli by directed evolution of the translation initiation region
- 2020
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859 .- 1475-2859. ; 19:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Recombinant proteins are often engineered with an N-terminal signal peptide, which facilitates their secretion to the oxidising environment of the periplasm (gram-negative bacteria) or the culture supernatant (gram-positive bacteria). A commonly encountered problem is that the signal peptide influences the synthesis and secretion of the recombinant protein in an unpredictable manner. A molecular understanding of this phenomenon is highly sought after, as it could lead to improved methods for producing recombinant proteins in bacterial cell factories.Results: Herein we demonstrate that signal peptides contribute to an unpredictable translation initiation region. A directed evolution approach that selects a new translation initiation region, whilst leaving the amino acid sequence of the signal peptide unchanged, can increase production levels of secreted recombinant proteins. The approach can increase production of single chain antibody fragments, hormones and other recombinant proteins in the periplasm of E. coli.Conclusions: The study demonstrates that signal peptide performance is coupled to the efficiency of the translation initiation region.
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| 7. |
- Mirzadeh, Kiavash, 1990-, et al.
(författare)
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Synthetically evolving translation initiation regions for protein production
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Bacteria are widely employed as cell factories to produce recombinant proteins that are used as biopharmaceuticals and industrial enzymes. Typically, a protein coding sequences is cloned into an expression vector that contains a number of genetic modules designed for high-level protein production, including those that allow efficient translation within the translation initiation region (TIR). Perplexingly, recombinant protein levels can vary in an unpredictable and context-dependent manner although all sequence features permitting maximum production levels are present. Here, we have taken a systematic approach to evaluate the efficiency of the TIR generated in the commonly used pET28a expression vector. By using a PCR-based randomisation approach that generates sequence variance covering the entire TIR, the most effective synthetically evolved TIRs within large TIR libraries were identified through a simple cell survival assay. This allowed us to determine which internal region of the TIR yields the most significant increase in protein production. Data presented in this study provide a framework for obtaining a synthetically evolved TIR that yields high protein production levels.
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