Integrative expression vectors for overexpression of xylitol dehydrogenase (XYL2) in Osmotolerant yeast, Candida glycerinogenes WL2002-5

Background:The oncoprotein binding (OPB) domain of Yin Yang 1 (YY1) consists of 26 amino acids between G201 and S226, and is involved in YY1 interaction with multiple oncogene products, including MDM2, AKT, EZH2 and E1A. Through the OPB domain, YY1 promotes the oncogenic or proliferative regulation of these oncoproteins in cancer cells. We previously demonstrated that a peptide with the OPB sequence blocked YY1-AKT interaction and inhibited breast cancer cell proliferation.Objective:In the current study, we characterized the OPB domain and determined a minimal region for peptide design to suppress cancer cellMethods:Using alanine-scan method, we identified that the amino acids at OPB C-terminal are essential to YY1 binding to AKT. Further studies suggested that serine and threonine residues, but not lysines, in OPB play a key role in YY1-AKT interaction. We generated GFP fusion expression vectors to express OPB peptides with serially deleted N-terminal and found that OPB1 (i.e. G201-S226) is cytoplasmic, but OPB2 (i.e. E206-S226), OPB3 (i.e. E206-S226) and control peptide were both nuclear and cytoplasmic.Results:Both OPB1 and 2 inhibited breast cancer cell proliferation and migration, but OPB3 exhibited similar effects to control. OPB1 and 2 caused cell cycle arrest at G1 phase, increased p53 and p21 expression, and reduced AKT(S473) phosphorylation in MCF-7 cells, but not in MDA-MB-231 cells.Conclusion:: Overall, the serines and threonines of OPB are essential to YY1 binding to oncoproteins, and OPB peptide can be minimized to E206-S226 that maintain inhibitory activity to YY1- promoted cell proliferation.

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A Productive Expression Platform Derived from Host-Restricted Eilat Virus: Its Extensive Validation and Novel Strategy

Viruses ◽

10.3390/v13040660 ◽

2021 ◽

Vol 13 (4) ◽

pp. 660

Author(s):

Lu Tan ◽

Yiwen Zhang ◽

Xingxing Wang ◽

Dal Young Kim

Keyword(s):

Expression Vectors ◽

Structural Genes ◽

Host Restriction ◽

Expression Platform ◽

Mosquito Cells ◽

Wide Range ◽

Restriction Property ◽

Infected Cells ◽

Multiple Levels ◽

Novel Strategy

Most alphaviruses are transmitted by mosquitoes and infect a wide range of insects and vertebrates. However, Eilat virus (EILV) is defective for infecting vertebrate cells at multiple levels of the viral life cycle. This host-restriction property renders EILV an attractive expression platform since it is not infectious for vertebrates and therefore provides a highly advantageous safety profile. Here, we investigated the feasibility of versatile EILV-based expression vectors. By replacing the structural genes of EILV with those of other alphaviruses, we generated seven different chimeras. These chimeras were readily rescued in the original mosquito cells and were able to reach high titers, suggesting that EILV is capable of packaging the structural proteins of different lineages. We also explored the ability of EILV to express authentic antigens via double subgenomic (SG) RNA vectors. Four foreign genetic materials of varied length were introduced into the EILV genome, and the expressed heterologous genetic materials were readily detected in the infected cells. By inserting an additional SG promoter into the chimera genome containing the structural genes of Chikungunya virus (CHIKV), we developed a bivalent vaccine candidate against CHIKV and Zika virus. These data demonstrate the outstanding compatibility of the EILV genome. The produced recombinants can be applied to vaccine and diagnostic tool development, but more investigations are required.

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Regulated overproduction of the GAL4 gene product greatly increases expression from galactose-inducible promoters on multi-copy expression vectors in yeast

Gene ◽

10.1016/0378-1119(87)90107-7 ◽

1987 ◽

Vol 61 (2) ◽

pp. 123-133 ◽

Cited By ~ 46

Author(s):

Loren D. Schultz ◽

Kathryn J. Hofmann ◽

Lawrence M. Mylin ◽

Donna L. Montgomery ◽

Ronald W. Ellis ◽

...

Keyword(s):

Gene Product ◽

Expression Vectors ◽

Inducible Promoters

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Duck RIG-I CARD Domain Induces the Chicken IFN-βby Activating NF-κB

BioMed Research International ◽

10.1155/2015/348792 ◽

2015 ◽

Vol 2015 ◽

pp. 1-6 ◽

Cited By ~ 1

Author(s):

Yang Chen ◽

Zhengyang Huang ◽

Bin Wang ◽

Qinming Yu ◽

Ran Liu ◽

...

Keyword(s):

Innate Immunity ◽

Eukaryotic Expression ◽

Poly I:C ◽

Expression Vectors ◽

Luciferase Reporter ◽

Enzyme Linked Immunosorbent Assay ◽

Regulatory Domains ◽

Polycytidylic Acid ◽

Card Domain ◽

Inducible Gene

Retinoic acid-inducible gene I- (RIG-I-) like receptors (RLRs) have recently been identified as cytoplasmic sensors for viral RNA. RIG-I, a member of RLRs family, plays an important role in innate immunity. Although previous investigations have proved that RIG-I is absent in chickens, it remains largely unknown whether the chicken can respond to RIG-I ligand. In this study, the eukaryotic expression vectors encoding duRIG-I full length (duck RIG-I, containing all domains), duRIG-I N-terminal (containing the two caspase activation and recruitment domain, CARDs), and duRIG-I C-terminal (containing helicase and regulatory domains) labeled with 6*His tags were constructed successfully and detected by western blotting. Luciferase reporter assay and enzyme-linked immunosorbent assay (ELISA) detected the duRIG-I significantly activated NF-κB and induced the expression of IFN-βwhen polyinosinic-polycytidylic acid (poly[I:C], synthetic double-stranded RNA) challenges chicken embryonic fibroblasts cells (DF1 cells), while the duRIG-I was inactive in the absence of poly[I:C]. Further analysis revealed that the CARDs (duRIG-I-N) induced IFN-βproduction regardless of the presence of poly[I:C], while the CARD-lacking duRIG-I (duRIG-I-C) was not capable of activating downstream signals. These results indicate that duRIG-I CARD domain plays an important role in the induction of IFN-βand provide a basis for further studying the function of RIG-I in avian innate immunity.

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