A simple procedure for expression and purification of selected non-structural (α and β) herpes simplex virus 1 (HSV-1) proteins

Košovský, Ján; Ďurmanová, Vladimı́ra; Kúdelová, M; Režuchová, Ingeborg; Tkáčiková, Ľudmila; Rajčáni, J

doi:10.1016/s0166-0934(00)00281-0

Cited by 3 publications

(4 citation statements)

References 28 publications

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“…In our study, a strong degradation was noticed in the proteins expressed using the PinPoint Xa-1 vector despite the presence of protease inhibitors. The production of untagged biotinylated proteins was also reported even after careful optimization of the culture conditions [ 52 , 53 ]. In contrast to the Nt region, the Ct region was well expressed using the two vectors.…”

Section: Discussionmentioning

confidence: 99%

“…Differential expression was noticed only with the BPT-Ct as JM109 E. coli cells exhibit a tenth less expressed protein than E. coli BL21 (DE3) (data not shown). As opposed to the GST-Ct and to the GST, we could not purify the BPT-Ct and the BPT by affinity chromatography using different protocols [ 52 , 54 ]. Surprisingly, despite the instability of the biotinylated fusion proteins, they have been found to be heat treatment resistant at 70°C for 5 minutes.…”

Section: Discussionmentioning

confidence: 99%

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Evaluation of an in silico predicted specific and immunogenic antigen from the OmcB protein for the serodiagnosis of Chlamydia trachomatis infections

et al. 2008

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Background: The OmcB protein is one of the most immunogenic proteins in C. trachomatis and C. pneumoniae infections. This protein is highly conserved leading to serum cross reactivity between the various chlamydial species. Since previous studies based on recombinant proteins failed to identify a species specific immune response against the OmcB protein, this study evaluated an in silico predicted specific and immunogenic antigen from the OmcB protein for the serodiagnosis of C. trachomatis infections.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Evaluation of an in silico predicted specific and immunogenic antigen from the OmcB protein for the serodiagnosis of Chlamydia trachomatis infections

et al. 2008

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“…The gD1 (US6) ectodomain (gD1/313) lacked the TM domain and the signal sequence (spanning from amino acids 26 to 340) but contained the major antigenic epitopes VII, II and XI. The IE fusion polypeptide ICP27/UL54 was prepared according to Košovský et al (2000). Briefly, each DNA fragment was double-digested with suitable restriction enzymes and inserted into the polylinker site of the pPPXa-1 expression vector (Promega), which encodes the biotinylated portion of fusion protein.…”

Section: Methodsmentioning

confidence: 99%

Immune response and cytokine production following immunization with experimental herpes simplex virus 1 (HSV-1) vaccines

et al. 2008

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Balb/c mice were immunized with the recombinant fusion protein gD1/313 (FpgD1/313 representing the ectodomain of HSV-1 gD), with the non-pathogenic ANGpath gE-del virus, with the plasmid pcDNA3.1-gD expressing full-length gD1 and with the recombinant immediate early (IE) HSV-1 protein ICP27. Specific antibodies against these antigens (as detected by ELISA) reached high titers with the exception of the DNA vaccine. High-grade protection against challenge with the virulent strain SC16 was found following immunization with the pcDNA3.1-gD plasmid and with the gE-del virus. Medium grade, but satisfactory protection developed after immunization with the FpgD1/313 and minimum grade protection was seen upon immunization with the IE/ICP27 polypeptide. A considerable response of peripheral blood cells (PBL) and splenocytes in the lymphocyte transformation test (LTT) was found in mice immunized with FpgD1/313, with the pcDNA3.1-gD plasmid and with the live ANGpathgE-del virus. For lymphocyte stimulation in vitro, the FpgD1/313 antigen was less effective than the purified gD1/313 polypeptide (cleaved off from the fusion protein); both proteins elicited higher proliferation at the 5 microg per 0.1 mL dose than at the 1 microg per 0.1 mL dose. The secretion of Th type 1 (TNF, IFN-gamma and IL-2) and Th type 2 (IL-4 and IL-6) cytokines was tested in the medium fluid of purified PBL and splenocyte cultures; their absolute values were expressed in relative indexes. The PBL from FpgD1/313 immunized mice showed increased secretion of both T(H)1 (TNF) as well as T(H)2 (IL-4) cytokines (7-10-fold, respectively). Splenocytes from FpgD1/313 immunized mice showed a significant (23-fold) increase in IL-4 production.

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“…This allows for an optimal recovery of these proteins at the purification stage. The following is a sample of papers that used this protocol to recover biotinylated proteins: [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25]. Following the pre-adsorption phase, the column was then washed with 16 ml of 10 % acetic acid and 0.1 M NaHPO 4 until the pH of the flow-through was >6.8.…”

Section: Biotin Avidin Columnmentioning

confidence: 99%

What maintains the high intra-follicular estradiol concentration in pre-ovulatory follicles?

Bentov

Jurisicova

Kenigsberg

et al. 2015

J Assist Reprod Genet

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Purpose The purpose of the study was to establish the mechanism by which the estrogen concentration difference between the follicular fluid and the serum is maintained. Methods We used dialysis membrane with a pore size of <3 KD to characterize the estrogen-binding capacity of the follicular fluid. We performed PCR, western blot, and ELISA on luteinized granulosa cells to determine if sex hormonebinding globulin (SHBG) is produced by granulosa cells, and finally we used affinity columns and mass spectrometry to identify the estrogen-binding protein in the follicular fluid. Results We found that a significant estrogen concentration difference is maintained in a cell-free system and is lost with proteolysis of the follicular fluid proteins. Luteinized granulosa cells are likely not a source of SHBG, as we were not able to detect expression of SHBG in these cells. Perlecan was the most highly enriched follicular fluid protein in the affinity columns. Conclusions We were able to identify perlecan as the most likely candidate for the major estrogen-binding protein in the follicular fluid.

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A simple procedure for expression and purification of selected non-structural (α and β) herpes simplex virus 1 (HSV-1) proteins

Cited by 3 publications

References 28 publications

Evaluation of an in silico predicted specific and immunogenic antigen from the OmcB protein for the serodiagnosis of Chlamydia trachomatis infections

Evaluation of an in silico predicted specific and immunogenic antigen from the OmcB protein for the serodiagnosis of Chlamydia trachomatis infections

Immune response and cytokine production following immunization with experimental herpes simplex virus 1 (HSV-1) vaccines

What maintains the high intra-follicular estradiol concentration in pre-ovulatory follicles?

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