Construction of a gene of the human tumor-associated antigen VNTR(MUC1) bound to streptavidin, its expression inEscherichia coli, and the study of properties of the hybrid protein

Gul’ko, L. B.; Павлова, О. В.; D’yakov, N. A.; Okorokova, N. A.; Ratmanova, K. I.; Логунова, Н. Н.; Bobreneva, R. A.; Макаров, Владимир Александрович; Yurin, V. L.; Вейко, В. П.; Дебабов, В. Г.

doi:10.1007/bf02758665

Cited by 3 publications

(5 citation statements)

References 28 publications

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“…It was demonstrated that the signal peptides of pro streptavi din from the S. avidinii and pro enterotoxins from the S. aureus are recognized by the Sec system of the E. coli cells, and thus both the efficient translocation of these pro polypeptides in the periplasmic space of cells and their full maturation are provided [7,9,19,20].…”

Section: Resultsmentioning

confidence: 99%

“…CIP106686, collection of microorganisms, Pasteur Institute, France) were used in the study. The plasmids containing pro enterotoxins B, H (SEB, SRH) and pro streptavidin (SAV) genes were previously constructed [7,9,18,19]. The pER plasmid containing the kanamycin resis tance gene and promoter-operator region of the udp gene from E. coli was previously constructed [12].…”

Section: Methodsmentioning

confidence: 99%

“…The interest in the study of the peculiarities of the functioning of Sec system pro tein translocation is determined by its practical impor tance. Post translational modifications of the proteins are performed in periplasmic space; therefore, removal of the recombinant protein to the periplasm allows a significant decrease in the effect of proteases, as well as a considerable simplification of its subse quent isolation and purification [6][7][8][9].…”

Section: Introductionmentioning

confidence: 99%

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Investigation of protein translocation Sec-system with heterologous gene expression in Shewanella oneidensis MR-1 bacterium cells

Mordkovich

Okorokova

Вейко

2015

Appl Biochem Microbiol

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A comparison of the primary structures of the protein translocation Sec system proteins in the Shewanella oneidensis MR 1 and Escherichia coli bacteria was carried out. The process of translocation of recombinant pro enterotoxins (SEB and SEH) from Staphylococcus aureus and pro streptavidin (SAV) from Streptomyces avidinii in the S. oneidensis MR 1 and E. coli cell periplasm was studied. It was demonstrated that these marker proteins are transferred into the periplasmic space of the S. oneidensis MR 1 transformant strain cells. The identity of N terminal amino acid sequences of mature recombinant SEB, SEH, and SAV proteins (generated during post translation proteolysis of leader peptide by the Sec system both in E. coli and S. oneidensis MR 1) was established.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Investigation of protein translocation Sec-system with heterologous gene expression in Shewanella oneidensis MR-1 bacterium cells

Mordkovich

Okorokova

Вейко

2015

Appl Biochem Microbiol

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“…A large number of the Ser and Thr residues that are supposed to carry oligosaccharides are located in the tandem repeat region (VNTR ), where each repeat contains 20 amino acid residues, including five potential Oglycosylation sites. Following the technique developed to construct fragments that encode different amounts of repeats from VNTR of human mucin MUC 1 [ 25 ], a fragment encoding 21 tandem repeats was obtained ( Fig. 2 ).…”

Section: Resultsmentioning

confidence: 99%

“…The Tr21 fragment was obtained using the technique developed to produce fragments containing different numbers of tandem repeats from the VNTR region of the human MUC 1 gene [ 25 ], and it was cloned into the vector pUC 18 – TR 21 (at the restriction sites HindIII and BamHI).…”

Section: Methodsmentioning

confidence: 99%

Cell Models for the Investigation of the Role of the Mucin MUC1 Extracellular Domain in Metastasizing

et al. 2014

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The speculations on the role of MUC1, a substance which is overexpressed in glandular cancer cells, on the metastatic potential of such cells are rooted in data that seem to indicate that cell malignization correlates with a change from the apical localization of mucin MUC1 to a peripheral one. Nonetheless, the role of MUC1 in cancer metastasizing remains far from clear. The major hurdle remains the absence of adequate cell models. The aim of the present study was to create cell models that present different fragments of the human mucin MUC1 extracellular domain on their surface. Genetic constructions were generated on the basis of the plasmid vector pEGFP-N3. These constructions contain fusion genes coding for chimeric proteins composed of different combinations of mucin MUC1 functional domains and identification markers (FLAG-epitope, located at the N-terminus, and EGFP, located at the C-terminus of the chimeric proteins). These constructions were used for a stable transformation of HT-29 human cancer cells. The transformants obtained were characterized by flow cytometry. The low expression level of endogenous mucin MUC1 and the high expression level of recombinant proteins were confirmed by real-time PCR. The microscopic examination of the transformed cells confirmed the membrane localization of the fusion proteins. The resulting cell models could be used to investigate the role of the mucin MUC1 domains in cancer cell metastasizing. The obtained cells are used as an applicable model of MUC1-expressing cancers and might be used to study the role of different functional fragments of mucin MUC1 in metastasizing.

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Uridine phosphorylase from Shewanella oneidensis MR-1: Heterological expression, regulation, transcription, and properties

Mordkovich

Manuvera

Вейко

et al. 2012

Appl Biochem Microbiol

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Construction of a gene of the human tumor-associated antigen VNTR(MUC1) bound to streptavidin, its expression inEscherichia coli, and the study of properties of the hybrid protein

Cited by 3 publications

References 28 publications

Investigation of protein translocation Sec-system with heterologous gene expression in Shewanella oneidensis MR-1 bacterium cells

Investigation of protein translocation Sec-system with heterologous gene expression in Shewanella oneidensis MR-1 bacterium cells

Cell Models for the Investigation of the Role of the Mucin MUC1 Extracellular Domain in Metastasizing

Uridine phosphorylase from Shewanella oneidensis MR-1: Heterological expression, regulation, transcription, and properties

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