Characterization of disulfide bonds in recombinant proteins: reduced human interleukin 2 in inclusion bodies and its oxidative refolding

Unique to aspartic proteinases from plants are the presence of approximately 100 amino acid regions, which are usually excised during activation of the zymogen. These sequences are termed ‘Plant-Specific Inserts’ and are implicated in membrane interactions of their parent enzymes, including vacuolar targeting and host defense. The need to further characterize the structure-function rol (s) of plant-specific inserts stimulated the current study of the characterization of Solanum tuberosum AP (StAP). Recombinant expression of wild-type StAP resulted in the 54 kDa protein being visualized by SDS-PAGE analysis with a protein yield of 0.03%. A protein purification factor could not be established since activation of the protein at pH 2.2, 3.0, 3.7 and 5.5 was not achieved as evidenced by a lack of change in band patterns on SDS-PAGE as well as acidification and hemoglobin degradation assays. To potentially improve enzyme folding and activation ability, two mutants, (1) lacking the pre-signal sequence and (2) lacking both the signal sequence and the prosegment, were designed, sub-cloned, and expressed. Both products proved to be insoluble and inactive. New constructs were designed for the expression of StAP inclusion bodies for insoluble expression and subsequent re-folding of the protein. Additionally, CysAla mutations for each PSI Cys residue were made to investigate the rol (s) of plant-specific insert disulfide bonds in plant aspartic proteinase enzyme folding and structure. All PSI cysteine mutations (eight mutants) were successfully created using QuickChange MutagenesisTM

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The Combined Effect of Weaning Stress and Immune Activation during Pig Gestation on Serum Cytokine and Analyte Concentrations

Animals ◽

10.3390/ani11082274 ◽

2021 ◽

Vol 11 (8) ◽

pp. 2274

Author(s):

Haley E. Rymut ◽

Laurie A. Rund ◽

Courtni R. Bolt ◽

Maria B. Villamil ◽

Bruce R. Southey ◽

...

Keyword(s):

Immune Activation ◽

Management Practices ◽

Interleukin 1 ◽

Interleukin 2 ◽

Blood Chemistry ◽

Respiratory Syndrome Virus ◽

Weaning Stress ◽

And Control ◽

Metabolic Hormone

Weaning stress can elicit changes in the metabolic, hormone and immune systems of pigs and interact with prolonged disruptions stemming from maternal immune activation (MIA) during gestation. The present study advances the characterization of the combined effects of weaning stress and MIA on blood chemistry, immune and hormone indicators that inform on the health of pigs. Three-week-old female and male offspring of control gilts or gilts infected with the porcine reproductive and respiratory syndrome virus were allocated to weaned or nursed groups. The anion gap and bilirubin profiles suggest that MIA enhances tolerance to the effects of weaning stress. Interleukin 1 beta and interleukin 2 were highest among weaned MIA females, and cortisol was higher among weaned relative to nursed pigs across sexes. Canonical discriminant analysis demonstrated that weaned and nursed pigs have distinct chemistry profiles, whereas MIA and control pigs have distinct cytokine profiles. The results from this study can guide management practices that recognize the effects of the interaction between MIA and weaning stress on the performance and health of pigs.

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Synthesis and Characterization of Store-Operated Calcium Entry Inhibitors Active in the Submicromolar Range

International Journal of Molecular Sciences ◽

10.3390/ijms21249777 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9777

Author(s):

Camille Le Guilcher ◽

Tomas Luyten ◽

Jan B. Parys ◽

Mathieu Pucheault ◽

Olivier Dellis

Keyword(s):

Cell Activation ◽

Interleukin 2 ◽

Calcium Entry ◽

Ip3 Receptors ◽

Excitable Cells ◽

Cellular Targets ◽

Entry Inhibitors ◽

The Past ◽

Store Operated Calcium Entry

The store-operated calcium entry, better known as SOCE, forms the main Ca2+ influx pathway in non-excitable cells, especially in leukocytes, where it is required for cell activation and the immune response. During the past decades, several inhibitors were developed, but they lack specificity or efficacy. From the non-specific SOCE inhibitor 2-aminoethyl diphenylborinate (2-APB), we synthetized 16 new analogues by replacing/modifying the phenyl groups. Among them, our compound P11 showed the best inhibitory capacity with a Ki ≈ 75 nM. Furthermore, below 1 µM, P11 was devoid of any inhibitory activity on the two other main cellular targets of 2-APB, the IP3 receptors, and the SERCA pumps. Interestingly, Jurkat T cells secrete interleukin-2 under phytohemagglutinin stimulation but undergo cell death and stop IL-2 synthesis when stimulated in the presence of increasing P11 concentrations. Thus, P11 could represent the first member of a new and potent family of immunosuppressors.

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Refolding, purification, and characterization of constitutive-active human-Smad8 produced as inclusion bodies in ClearColi® BL21 (DE3)

Protein Expression and Purification ◽

10.1016/j.pep.2021.105878 ◽

2021 ◽

Vol 184 ◽

pp. 105878

Author(s):

Carla Lizbeth Segovia-Trinidad ◽

Bastian Quaas ◽

Zhaopeng Li ◽

Antonina Lavrentieva ◽

Yvonne Roger ◽

...

Keyword(s):

Inclusion Bodies ◽

Purification And Characterization

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Characterization of a Dye-Decolorizing Peroxidase from Irpex lacteus Expressed in Escherichia coli: An Enzyme with Wide Substrate Specificity Able to Transform Lignosulfonates

Journal of Fungi ◽

10.3390/jof7050325 ◽

2021 ◽

Vol 7 (5) ◽

pp. 325

Author(s):

Laura Isabel de de Eugenio ◽

Rosa Peces-Pérez ◽

Dolores Linde ◽

Alicia Prieto ◽

Jorge Barriuso ◽

...

Keyword(s):

Escherichia Coli ◽

Inclusion Bodies ◽

Veratryl Alcohol ◽

Dye Decolorization ◽

Irpex Lacteus ◽

Type D ◽

Lignin Peroxidases

A dye-decolorizing peroxidase (DyP) from Irpex lacteus was cloned and heterologously expressed as inclusion bodies in Escherichia coli. The protein was purified in one chromatographic step after its in vitro activation. It was active on ABTS, 2,6-dimethoxyphenol (DMP), and anthraquinoid and azo dyes as reported for other fungal DyPs, but it was also able to oxidize Mn2+ (as manganese peroxidases and versatile peroxidases) and veratryl alcohol (VA) (as lignin peroxidases and versatile peroxidases). This corroborated that I. lacteus DyPs are the only enzymes able to oxidize high redox potential dyes, VA and Mn+2. Phylogenetic analysis grouped this enzyme with other type D-DyPs from basidiomycetes. In addition to its interest for dye decolorization, the results of the transformation of softwood and hardwood lignosulfonates suggest a putative biological role of this enzyme in the degradation of phenolic lignin.

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