scholarly journals Biosynthesis of recombinant human pro-α1(III) chains in a baculovirus expression system: production of disulphide-bonded and non-disulphide-bonded species containing full-length triple helices

1995 ◽  
Vol 312 (3) ◽  
pp. 847-853 ◽  
Author(s):  
M Tomita ◽  
N Ohkura ◽  
M Ito ◽  
T Kato ◽  
P M Royce ◽  
...  
Keyword(s):  
Insect Cells ◽  
Incubation Temperature ◽  
Expression System ◽  
Significant Proportion ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Dermal Fibroblasts ◽  
Full Length ◽  
Type Iii Collagen ◽  
Procollagen Iii

We have investigated the expression of human procollagen III by insect cells infected with a recombinant baculovirus carrying cDNA for the pro-alpha1(III) chain of type-III collagen. A high level of expression was obtained, and a small proportion of the heterologously expressed pro-alpha1(III) chains formed normally disulphide-bonded procollagen III, which was secreted into the culture medium. This species displayed a melting temperature (Tm) of approx. 38 degrees C as assessed by its resistance to digestion by a mixture of trypsin and chymotrypsin, slightly lower than that of 39.5 degrees C for procollagen III synthesized by cultured human dermal fibroblasts, and reflected a slight degree of under-hydroxylation of prolyl residues. This is possibly a consequence of the lower incubation temperature of insect cells, or of an insufficiency of prolyl hydroxylase activity within them. A significant proportion of the expressed chains formed trimeric molecules of similar thermal stability containing an apparently full-length triple-helical region, but were not disulphide-bonded and not secreted. In addition to providing a source of recombinant human procollagen III, the system promises to be useful in the study of procollagen chain association and subsequent folding.

Production of recombinant androgen receptor in a heterologous expression system

1993 ◽  
Vol 39 (2) ◽  
pp. 346-352 ◽  
Author(s):  
O A Jänne ◽  
J J Palvimo ◽  
P Kallio ◽  
M Mehto ◽  
Y B Xie ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Binding Activity ◽  
Full Length ◽  
Biologically Active ◽  
Receptor Protein ◽  
Cell Protein ◽  
Domain Specific

Abstract To facilitate detailed studies of androgen receptor, we have produced a full-length receptor protein and some of its deletion mutants in Spodoptera frugiperda (Sf9) insect cells, using the baculovirus expression system. Recombinant baculovirus DNA-infected Sf9 cells expressed these proteins in very high quantities, which represented as much as 30-40% of total insect cell protein at 72 h after infection. Only < 1% of the recombinant protein was soluble in low-salt buffers; the majority formed electron-dense cytoplasmic aggregates 30-40 nm in diameter. These aggregates could be solubilized in 6 mol/L guanidine HCl, and biologically active receptor was generated by diluting the guanidine HCl preparation 20- to 50-fold. The full-length receptor, expressed either in a soluble or aggregated form, had characteristics typical of a native receptor: it bound steroids with high affinity and specificity, interacted with DNA in a sequence-specific fashion, and was recognized by domain-specific receptor antibodies. Androgen-receptor protein purified to homogeneity in guanidine HCl required the presence of Zn2+ ions during the refolding to reconstitute its DNA-binding form; ZnCl2 was not, however, needed to restore the receptor's steroid-binding activity.

scholarly journals Expression, Purification and Sublocalization of SARS-CoV Nucleocapsid Protein in Insect Cells

2004 ◽  
Vol 36 (11) ◽  
pp. 754-758 ◽  
Author(s):  
Ai-Xia Ren ◽  
You-Hua Xie ◽  
Yu-Ying Kong ◽  
Guan-Zhen Yang ◽  
Yao-Zhou Zhang ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Trichoplusia Ni ◽  
Insect Cells ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Functional Study ◽  
Expression And Purification ◽  
N Protein ◽  
Baculovirus Expression

Abstract The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, this report describe the expression and purification of recombinant SARS-CoV N protein using the baculovirus expression system. Recombinant hexa-histidine-tagged N protein with a molecular mass of 47 kD was produced in insect cells. Recombinant N protein was purified to near homogeneity by Ni2+-NTA affinity chromatography. In addition, we examined the subcellular localization of the N protein by confocal microscopy in Trichoplusia ni BT1 Tn 5B1–4 cells infected with recombinant baculovirus. The N protein was found localized in the cytoplasm as well as in the nucleolus. The purified recombinant N protein can be used in further functional study of SARS-CoV.

scholarly journals High-efficiency expression and characterization of the synaptic-vesicle monoamine transporter from baculovirus-infected insect cells

1998 ◽  
Vol 330 (2) ◽  
pp. 959-966 ◽  
Author(s):  
K. Michael SIEVERT ◽  
S. David THIRIOT ◽  
H. Robert EDWARDS ◽  
E. Arnold RUOHO
Keyword(s):  
Synaptic Vesicle ◽  
High Efficiency ◽  
Insect Cells ◽  
Expression System ◽  
Broad Band ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Sf9 Cells ◽  
Monoamine Transporter ◽  
Infected Cells

The full-length cDNA for the rat synaptic-vesicle monoamine transporter (VMAT2) containing a C-terminal polyhistidine epitope has been engineered into baculovirus DNA for expression in Spodoptera frugiperda (Sf9) insect cells. Using this recombinant baculovirus and cultured Sf9 cells, rVMAT2 has been expressed at levels of 7.8×106 transporters per cell, as assessed by [3H]dihydrotetrabenazine binding. A 1 l culture of infected cells produced approx. 15 nmol (900 μg) of transporter. rVMAT2 expressed in the Sf9 cells bound [3H]dihydrotetrabenazine with a KD of 31.2 nM and a Bmax of 19.9 pmol/mg. Two polypeptides of 55 and 63 kDa were identified using the photolabel, 7-azido-8-[125I]iodoketanserin ([125I]AZIK). Photoaffinity labelling of rVMAT2 by 1 nM [125I]AZIK was protectable by 10 μM tetrabenazine and 10 μM 7-aminoketanserin. Digitonin-solubilized VMAT2 was purified to greater than 95% homogeneity using immobilized Ni2+-affinity chromatography, followed by lectin (Concanavalin A) chromatography. The purified transporter migrates as a single broad band with a molecular mass of approx. 63 kDa, as analyzed by SDS/PAGE. The purified transporter retained the ability to bind ligands ([125I]AZIK and [3H]dihydrotetrabenazine). The purified VMAT2 bound [3H]dihydrotetrabenazine with a KD of 86.2 nM. As is the case with the monoamine transporter from bovine chromaffin granule membranes, purified VMAT2 is covalently modified by dicyclohexylcarbodi-imide (DCCD) and is specifically labelled by [14C]DCCD. This labelling is inhibited by tetrabenazine and ketanserin. These data indicate that VMAT2 can be overexpressed using the baculovirus expression system and purified.

scholarly journals Expression of Helicobacter pylori ggt Gene in Baculovirus Expression System and Activity Analysis of its Products

2011 ◽  
Vol 60 (3) ◽  
pp. 203-207 ◽  
Author(s):  
MEI KONG ◽  
MING XU ◽  
YA-LONG HE ◽  
YOU-LI ZHANG
Keyword(s):  
Helicobacter Pylori ◽  
Insect Cells ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Gastric Carcinogenesis ◽  
Full Length ◽  
Functional Assay ◽  
Reading Frame ◽  
Baculovirus Expression ◽  
Insight Into

The gamma-glutamyltranspeptidase (GGT) of Helicobacter pylori (HpGT) is a newly found virulence factor. In an approach to gain insight into the gene function, the four domains of the HpGT were cloned and expressed in baculovirus expression system. The results of a functional assay showed that the HpGT products acted as GGT, even when the N-terminal 380 amino acids were deleted. However, only the full length open reading frame (ORF) of the HpGT gene was apparently effective on cell growth. This result indicated that the products of the full length ORF might have an important role in gastric carcinogenesis. In this paper, we are the first to report that changes of mitochondrial membrane potential can be detected using 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazole carbocyanine iodide (JC-1) staining in insect cells.

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

2018 ◽  
Vol 9 (03) ◽  
pp. 20204-20223
Author(s):  
Maghsoudi, Hossein ◽  
U Pati
Keyword(s):  
Dna Binding ◽  
P53 Protein ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Cross Linking ◽  
Mobility Shift ◽  
Dna Complexes ◽  
P53 Antibodies

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

scholarly journals Production, processing and partial purification of functional G protein βγ subunits in baculovirus-infected insect cells

1992 ◽  
Vol 286 (3) ◽  
pp. 677-680 ◽  
Author(s):  
J D Robishaw ◽  
V K Kalman ◽  
K L Proulx
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Insect Cells ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Partial Purification ◽  
Baculovirus Expression ◽  
Gamma Subunits ◽  
Infected Insect ◽  
Beta 2

As a result of the inability to resolve the heterogeneous mixture of G protein beta gamma subunits present in tissues, it has not been possible to compare different beta gamma subunits of the G proteins in terms of their proposed roles in receptor-effector coupling. This study was undertaken to establish the utility of the baculovirus expression system in producing homogeneous beta gamma subunits of defined composition for the comparative analysis of these subunits in reconstitution systems. In this study we report the expression, and appropriate post-translational processing, of recombinant beta 2, gamma 2 and gamma 3 subunits. In addition, we show that the recombinant beta gamma subunits can be readily purified, and can functionally interact with the alpha subunits of the G proteins.

scholarly journals A Newly Designed EGFP-2A Peptide Monocistronic Baculoviral Vector for Concatenating the Expression of Recombinant Proteins in Insect Cells

Processes ◽  
2019 ◽  
Vol 7 (5) ◽  
pp. 291 ◽  
Author(s):  
Chih-Yu Wu ◽  
Chao-Wei Huang ◽  
Yu-Shin Nai ◽  
Pei-Yu Chu ◽  
Chung-Hsiung Wang ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Fluorescent Protein ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
New Combination ◽  
Vector System ◽  
Baculovirus Expression Vector

Recombinant proteins produced by the baculovirus expression vector system (BVES) have been widely applied in the agricultural and medical fields. However, the procedure for protein expression is inefficient and needs to be improved. Herein, we propose a simple construct that incorporates a selectable marker (enhanced green fluorescent protein, EGFP) and a picorna viral-derived “self-cleaving” 2A-like peptide to separate the EGFP and target proteins in a monocistronic baculovirus vector to facilitate isolation of the recombinant baculovirus in the BVES. In this study, porcine adiponectin (ADN), a secreted, multimeric protein with insulin-sensitizing properties, was used to demonstrate its utility in our EGFP-2A-based expression system. EGFP and ADN were simultaneously expressed by a recombinant alphabaculovirus. Co-expression of EGFP facilitates the manipulation of the following processes, such as determining expression kinetics and harvesting ADN. The results showed that the 2A “self-cleaving” process does not interfere with EGFP activity or with signal peptide removal and the secretion of recombinant ADN. Posttranslational modifications, including glycosylation, of the recombinant ADN occurred in insect cells, and the formation of various multimers was further verified. Most importantly, the insect-produced ADN showed a similar bioactivity to that of mammalian cells. This concept provides a practical and economic approach that utilizes a new combination of alphabaculovirus/insect cell expression systems for future applications.

scholarly journals Partial characterization of natural and recombinant human soluble CD23

1992 ◽  
Vol 286 (3) ◽  
pp. 819-824 ◽  
Author(s):  
K Rose ◽  
G Turcatti ◽  
P Graber ◽  
S Pochon ◽  
P O Regamey ◽  
...  
Keyword(s):  
Insect Cells ◽  
Peptide Mapping ◽  
Sequence Similarity ◽  
Expression System ◽  
Protein A ◽  
Baculovirus Expression System ◽  
Partial Characterization ◽  
Disulphide Bonds ◽  
Baculovirus Expression

The purification to homogeneity of an active soluble 25 kDa fragment of CD23, produced in insect cells using the baculovirus expression system, is described. Peptide mapping and analysis by Edman degradation and mass spectrometry permitted partial characterization of the protein. A total of 165 out of 172 residues, including N-terminal and C-terminal regions, were mapped. The positions of the two disulphide bonds in the IgE-binding region were also determined: residue 110 is joined to residue 124, and residue 42 to residue 133. Natural CD23 25 kDa fragment was also analysed and found to possess the same disulphide bond arrangement. These results extend the previously noted sequence similarity with lectins to elements of secondary structure.

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