scholarly journals Expression and Purification of a Mammalian Protein: Cytosolic Domain of IRE1α from Insect Sf21 Cells

2019 ◽  
Author(s):  
Amrita Oak ◽  
Grace Jansen ◽  
Christina Chan
Keyword(s):  
Spodoptera Frugiperda ◽  
Shuttle Vector ◽  
Expression System ◽  
Cell System ◽  
Dual Function ◽  
Xbp1 Mrna ◽  
Eukaryotic Proteins ◽  
Cytosolic Domain ◽  
Recombinant Bacmid ◽  
Mammalian Protein

AbstractEukaryotic proteins can be expressed in different heterologous systems. However, mammalian proteins in general have specific post-translational processing requirements that may not be fulfilled by a regular bacterial expression system. In this study, we use an insect cell system to express a mammalian protein of interest. Spodoptera frugiperda (Sf21) cells were used in conjunction with a baculoviral expression system to produce the cytosolic domain (CD) of IRE1, an endoplasmic reticulum (ER) stress sensor protein. Inositol Requiring Enzyme 1 (IRE1) is a dual function kinase and endoribonuclease protein that cleaves X-box binding protein (XBP1) mRNA. We used the pFastBac plasmid to insert the coding sequence into a recombinant bacmid shuttle vector which was then used to infect Sf21 cells. The expressed protein was then purified with an MBPTrap column to obtain >85% pure protein.

High Level Expression of Active Human Prothrombin in a Vaccina Virus Expression System

1992 ◽  
Vol 68 (02) ◽  
pp. 119-124 ◽  
Author(s):  
F G Falkner ◽  
P L Turecek ◽  
R T A MacGillivray ◽  
W Bodemer ◽  
F Scheiflinger ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Cell Lines ◽  
Expression System ◽  
Human Cell Line ◽  
Cell System ◽  
Time Saving ◽  
Expression Levels ◽  
Mammalian Cell Lines ◽  
Cell Line Hela ◽  
Virus Expression

SummaryWe have worked out an efficient and time saving procedure for the expression of recombinant human prothrombin. The glycoprotein was expressed in the vaccinia virus expression system in several mammalian cell lines. The kidney cell lines Vero and BHK and the human cell line Hela were found to efficiently secrete prothrombin. Expression levels of 3–4 µg of factor II per 106 cells per day corresponding to 18–23 mU per 106 cells per day were achieved. Since the expression levels obtained with the vaccinia virus/Vero cell system were comparable to those obtained in amplified transformed CHO cells it provides an alternative system for the efficient expression of human prothrombin and may allow to further elucidate structure-function relationships of (pro)thrombin and its various effectors.

Activation of recombinant trp by thapsigargin in Sf9 insect cells

1994 ◽  
Vol 267 (5) ◽  
pp. C1501-C1505 ◽  
Author(s):  
L. Vaca ◽  
W. G. Sinkins ◽  
Y. Hu ◽  
D. L. Kunze ◽  
W. P. Schilling
Keyword(s):  
Expression System ◽  
Cation Selectivity ◽  
Drosophila Protein ◽  
Insect Cell Expression System ◽  
Cell Expression ◽  
Insect Cell Expression ◽  
Mammalian Protein ◽  
Entry Mechanism ◽  
Proline Rich Region ◽  
Cell Expression System

The mammalian protein responsible for Ca2+ release-activated current (Icrac) may be homologous to the Drosophila protein designated trp. Thus the activity of trp, and another Drosophila protein designated trp-like or trpl, may be linked to depletion of the internal Ca2+ store via the so-called capacitative Ca2+ entry mechanism. To test this hypothesis, the effect of thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca2+ pump, on trp- and trpl-induced whole cell membrane current was determined using the baculovirus Sf9 insect cell expression system. The results demonstrate that trp and trpl form Ca(2+)-permeable cation channels. The trpl encodes a nonselective cation channel that is constitutively active under basal nonstimulated conditions and is unaffected by thapsigargin, whereas trp is more selective for Ca2+ than Na+ and is activated by depletion of the internal Ca2+ store. Although evaluation of cation selectivity suggests that trp is not identical to the channel responsible for Icrac, these channels must share some structural feature(s) since both are activated by thapsigargin. A unique proline-rich region in the COOH-terminal tail of trp, which is absent in trpl, may be necessary for capacitative Ca2+ entry.

scholarly journals Development of a High-Throughput Cell-Based Reporter Assay to Identify Stabilizers of Tumor Suppressor Pdcd4

2009 ◽  
Vol 15 (1) ◽  
pp. 21-29 ◽  
Author(s):  
Johanna S. Blees ◽  
Tobias Schmid ◽  
Cheryl L. Thomas ◽  
Alyson R. Baker ◽  
Lauren Benson ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
High Throughput ◽  
Expression System ◽  
Control Cell ◽  
Cell System ◽  
Luciferase Expression ◽  
The Novel ◽  
Pure Compounds ◽  
The Stability

The novel tumor suppressor Pdcd4 affects tumorigenesis by inhibiting translation. Pdcd4 is phosphorylated and subsequently lost by proteasomal degradation in response to tumor-promoting conditions. Here, the authors describe the development of a reporter cell system to monitor the stability of Pdcd4. The phosphorylation-dependent degradation domain (“target”) or an adjacent (“off-target”) region of Pdcd4 was cloned into a luciferase expression system. The target constructs were responsive to Pdcd4 degrading conditions (e.g., TPA, p70S6K1 overactivation), whereas the off-target constructs remained stable. The system was optimized for and shown to be reliable in a high-throughput compatible 384-well format. Screening of 15,275 pure compounds resulted in a hit rate of 0.30% (>" xbd="1092" xhg="1069" ybd="1185" yhg="1147"/>50% inhibition of TPA-induced loss of signal, confirmed by reassay). Among the hits were inhibitors of previously identified critical signaling events for TPA-induced Pdcd4 degradation. One compound was identified to be nonspecific using the off-target control cell line. Screening of 135,678 natural product extracts yielded 42 confirmed, specific hits. Z′ averaged 0.58 across 446 plates. Further characterization of active natural products and synthetic compounds is expected to identify novel Pdcd4 stabilizers that may be useful in targeting translation to prevent or treat cancers. (Journal of Biomolecular Screening 2010:21-29)

Expression of Winged Bean Basic Agglutinin in Spodoptera frugiperda Insect Cell Expression System

2001 ◽  
Vol 21 (3) ◽  
pp. 361-367 ◽  
Author(s):  
V. R. Srinivas ◽  
Kiran Bachhawat-Sikder ◽  
Saman Habib ◽  
Seyed E. Hasnain ◽  
Avadhesha Surolia
Keyword(s):  
Spodoptera Frugiperda ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Winged Bean ◽  
Carbohydrate Binding ◽  
Binding Properties ◽  
Insect Cell Expression System ◽  
Cell Expression ◽  
Insect Cell Expression ◽  
Cell Expression System

In this paper we report the successful expression of the winged bean basic agglutinin (WBA I) in insect cells infected with a recombinant baculovirus carrying the WBA I gene and its characterization in terms of its carbohydrate binding properties. The expressed protein appears to have a lower molecular weight than the native counterpart which is consistent with the lack of glycosylation of the former. Moreover, the expressed protein maintains its dimeric nature. Hence, a role for glycosylation in modulation of dimerization of WBA I is ruled out unlike Erythrina corallodendron (EcorL). Despite this the protein is active, with its sugar specificity unaltered.

Dictyostelium discoideum —a promising expression system for the production of eukaryotic proteins

2008 ◽  
Vol 22 (12) ◽  
pp. 4055-4066 ◽  
Author(s):  
Ranjana Arya ◽  
Alok Bhattacharya ◽  
Kulvinder Singh Saini
Keyword(s):  
Dictyostelium Discoideum ◽  
Expression System ◽  
Eukaryotic Proteins

scholarly journals Development of Inducible Systems To Engineer Conditional Mutants of Essential Genes of Helicobacter pylori

2008 ◽  
Vol 74 (7) ◽  
pp. 2095-2102 ◽  
Author(s):  
Ivo G. Boneca ◽  
Chantal Ecobichon ◽  
Catherine Chaput ◽  
Aurélie Mathieu ◽  
Stéphanie Guadagnini ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Shuttle Vector ◽  
Expression System ◽  
Gene Promoter ◽  
Start Codon ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Kanamycin Cassette ◽  
H Pylori

ABSTRACT The Escherichia coli-Helicobacter pylori shuttle vector pHeL2 was modified to introduce the inducible LacIq-pTac system of E. coli, in which the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacIq repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring β-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in H. pylori. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential pbp1 and ftsI genes encoding penicillin-binding proteins 1 and 3 of H. pylori, respectively. Both genes were cloned into pILL2150 and introduced in the parental H. pylori strain N6. The chromosomally harbored pbp1 and ftsI genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-β-d-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of H. pylori. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of H. pylori isolates with different genetic backgrounds.

scholarly journals GPCR-Based Bioactive Peptide Screening Using Phage-Displayed Peptides and an Insect Cell System for Insecticide Discovery

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 583
Author(s):  
Man-Yeon Choi ◽  
Robert K. Vander Meer
Keyword(s):  
Pest Management ◽  
Insect Cell ◽  
Bioactive Peptides ◽  
Insect Pest ◽  
Expression System ◽  
Management Strategies ◽  
Screening Method ◽  
Risk Process ◽  
Cell System ◽  
Bioactive Peptide

The discovery of new insecticides improves integrated pest management (IPM), but is usually a long high-risk process with a low probability of success. For over two decades, insect neuropeptides (NPs) and their G-protein coupled receptors (GPCRs) have been considered as biological targets for insect pest control, because they are involved in almost all physiological processes associated with insect life stages. A key roadblock to success has been the question of how large volume chemical libraries can be efficiently screened for active compounds. New genomic and proteomic tools have advanced and facilitated the development of new approaches to insecticide discovery. In this study, we report a novel GPCR-based screening technology that uses millions of short peptides randomly generated by bacteriophages, and a method using an insect Sf9 cell expression system. The fire ant is a good model system, since bioactive peptides have been identified for a specific GPCR. The novel small peptides could interfere with the target GPCR-ligand functions. Therefore, we refer to this new mechanism as “receptor interference” (RECEPTORi). The GPCR-based bioactive peptide screening method offers multiple advantages. Libraries of phage-displayed peptides (~109 peptides) are inexpensive. An insect cell-based screening system rapidly leads to target specific GPCR agonists or antagonists in weeks. Delivery of bioactive peptides to target pests can be flexible, such as topical, ingestion, and plant-incorporated protectants. A variety of GPCR targets are available, thus minimizing the development of potential insecticide resistance. This report provides the first proof-of-concept for the development of novel arthropod pest management strategies using neuropeptides, and GPCRs.

Expression of H5N1Avian influenza virushaemagglutinin in a baculovirus expression system

2007 ◽  
Vol 4 (3) ◽  
pp. 233-237 ◽  
Author(s):  
Dun Jifeng ◽  
Pu Juan ◽  
Zhou Yingchun ◽  
Liu Jinhua
Keyword(s):  
Shuttle Vector ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Sf9 Cells ◽  
Ha Gene ◽  
Sds Page ◽  
Reaction Specificity ◽  
H5 Subtype ◽  
Ha Protein

AbstractThe haemagglutinin (HA) gene of H5N1Avian influenza virus(AIV) was amplified from the plasmid pGEM-HA and cloned into the baculovirus transfer plasmid pFastBacHT to construct the recombinant transfer vector pFastBacHT-HA. The pFastBacHT-HA was transformed into DH10Bac competent cells, transposed with a shuttle vector (Bacmid) and the transposition rBacmid-HA was constructed. The recombinant baculovirus was harvested from sf9 cells transfected with rBacmid-HA. The expressed HA protein was identified and analysed by SDS–PAGE, Western blot and haemadsorption assay. The 66 kDa protein could only react with chicken serum positive to H5 subtype AIV. The haemadsorption assay showed that the sf9 cells infected with rBacmid-HA baculovirus could absorb chicken red blood cells. These results indicated that the HA protein was successfully expressed in sf9 cells, with reaction specificity to H5 subtype antiserum.

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