scholarly journals Large-scale production and purification of functional recombinant bovine rhodopsin with the use of the baculovirus expression system

1999 ◽  
Vol 342 (2) ◽  
pp. 293-300 ◽  
Author(s):  
Corné H. W. KLAASSEN ◽  
Petra H. M. BOVEE-GEURTS ◽  
Godelieve L. J. DECALUWÉ ◽  
Willem J. DEGRIP
Keyword(s):  
Membrane Proteins ◽  
Large Scale ◽  
Structural Changes ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Scale Production ◽  
Protein Activation ◽  
Bovine Rhodopsin ◽  
Lipid Environment

Here we describe a generic procedure for the expression and purification of milligram quantities of functional recombinant eukaryotic integral membrane proteins, exemplified by hexahistidine-tagged bovine rhodopsin. These quantities were obtained with the recombinant baculovirus/Sf9 insect cell-based expression system in large-scale bioreactor cultures with the use of a serum-free and protein-free growth medium. After optimization procedures, expression levels up to 4 mg/l were established. The recombinant rhodopsin could be purified with high overall yield by using immobilized-metal-affinity chromatography on Ni2+-agarose. After reconstitution into a native lipid environment, the purified protein was functionally indistinguishable from native rhodopsin with regard to the following parameters: spectral absorbance band, structural changes after photoactivation, and G-protein activation. The procedures developed can be adapted to other membrane proteins. The ability to produce and purify tens of milligrams of functional recombinant eukaryotic membrane protein meets the ever-increasing demand of material necessary to perform detailed biochemical and structural biophysical studies that are essential in unravelling their working mechanism at a molecular level.

scholarly journals Large-scale production and purification of the human green cone pigment: characterization of late photo-intermediates

1998 ◽  
Vol 330 (3) ◽  
pp. 1201-1208 ◽  
Author(s):  
Peter M. A. M. VISSERS ◽  
Petra H. M. BOVEE-GEURTS ◽  
M. Daniël PORTIER ◽  
Corné H. W. KLAASSEN ◽  
Willem J. DEGRIP
Keyword(s):  
Large Scale ◽  
Ph Dependence ◽  
Spectral Characteristics ◽  
Recombinant Baculovirus ◽  
Single Step ◽  
Scale Production ◽  
Spectral Position ◽  
Cone Pigment ◽  
Lipid Environment

We present the first characterization of the late photo-intermediates (Meta I, Meta II and Meta III) of a vertebrate cone pigment in a lipid environment. Marked differences from the same pathway in the rod pigment were observed. The histidine-tagged human green cone pigment was functionally expressed in large-scale suspension cultures in Sf9 insect cells using recombinant baculovirus. The recombinant pigment was extensively purified in a single step by immobilized metal affinity chromatography and displays the expected spectral characteristics. The purified pigment was able to activate the rod G-protein transducin at about half the rate of the rod pigment. Following reconstitution into bovine retina lipid proteoliposomes, identification and analysis of the photo-intermediates Meta I, Meta II and Meta III was accomplished. Similar to the rod pigment, our results indicate the existence of a Meta I-Meta II equilibrium, but we find no evidence for pH dependence. Replacement of native Cl- by NO3- in the anion-binding site of the cone pigment affected the spectral position of the pigment itself and of the Meta I intermediate, but not that of Meta II and Meta III. The decay rate of the ‘active’ intermediate Meta II did not differ for the Cl- and NO3- state. However, in qualitative agreement with results reported before for chicken cone pigments, the rate of Meta II decay was significantly higher in the human cone pigment than in the rod pigment.

scholarly journals Large-Scale, pH-Dependent, Quaternary Structure Changes in an RNA Virus Capsid Are Reversible in the Absence of Subunit Autoproteolysis

2002 ◽  
Vol 76 (19) ◽  
pp. 9972-9980 ◽  
Author(s):  
Derek J. Taylor ◽  
Neel K. Krishna ◽  
Mary A. Canady ◽  
Anette Schneemann ◽  
John E. Johnson
Keyword(s):  
Coat Protein ◽  
Conformational Change ◽  
Conformational Changes ◽  
Large Scale ◽  
Quaternary Structure ◽  
Rna Virus ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Ph Dependent

ABSTRACT The assembly and maturation of the coat protein of a T=4, nonenveloped, single-stranded RNA virus, Nudaurelia capensis ω virus (NωV), was examined by using a recombinant baculovirus expression system. At pH 7.6, the coat protein assembles into a stable particle called the procapsid, which is 450 Å in diameter and porous. Lowering the pH to 5.0 leads to a concerted reorganization of the subunits into a 410-Å-diameter particle called the capsid, which has no obvious pores. This conformational change is rapid but reversible until slow, autoproteolytic cleavage occurs in at least 15% of the subunits at the lower pH. In this report, we show that expression of subunits with replacement of Asn-570, which is at the cleavage site, with Thr results in assembly of particles with expected morphology but that are cleavage defective. The conformational change from procapsid to capsid is reversible in N570T mutant virus-like particles, in contrast to wild-type particles, which are locked into the capsid conformation after cleavage of the coat protein. The reexpanded procapsids display slightly different properties than the original procapsid, suggesting hysteretic effects. Because of the stability of the procapsid under near-neutral conditions and the reversible properties of the cleavage-defective mutant, NωV provides an excellent model for the study of pH-induced conformational changes in macromolecular assemblies. Here, we identify the relationship between cleavage and the conformational change and propose a pH-dependent helix-coil transition that may be responsible for the structural rearrangement in NωV.

High-level expression of recombinant porcine LH receptor in baculovirus-infected insect cells or caterpillars

1995 ◽  
Vol 14 (1) ◽  
pp. 51-66 ◽  
Author(s):  
E Pajot-Augy ◽  
L Couture ◽  
V Bozon ◽  
J-J Remy ◽  
G Biache ◽  
...  
Keyword(s):  
Total Protein ◽  
Large Scale ◽  
Insect Cells ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Multiplicity Of Infection ◽  
Scale Production ◽  
Lh Receptor ◽  
Recombinant Receptor ◽  
Infected Cells

ABSTRACT Porcine LH receptor ectodomain was overexpressed in insect cells and lepidopteran larvae using the recombinant baculovirus expression system. A low multiplicity of infection yielded the largest active production, of approximately 107 receptors/cell or 3 μg active receptor/mg total protein in infected cells. The truncated ectodomain solubilized with Triton X-100 bound its ligand with a high affinity which was comparable with that of the native membrane receptor. Increasing the multiplicity of infection resulted in an optimum protein production of 0·6 mg receptor/mg total protein in infected cells. This receptor was largely inactive, probably trapped within aggregation pools. Active receptor could be recovered by dilution of the samples. No secretion of recombinant receptor was ever observed whatever the conditions of infection. Expression of the recombinant receptor in insect larvae was also tested. This low-cost system failed both to increase the amount of active receptor and to induce secretion into the haemolymph. Two methods remain for producing sizeable amounts of active receptor with this baculovirus/insect cell system. One relies on immunoaffinity purification of the active protein and requires large-scale production, and the other is based on the purification of overexpressed inactive receptor followed by renaturation.

scholarly journals Point mutations in bovine opsin can be classified in four groups with respect to their effect on the biosynthetic pathway of opsin

1996 ◽  
Vol 320 (3) ◽  
pp. 807-815 ◽  
Author(s):  
Godelieve L. J. DeCALUWÉ ◽  
Willem J. DeGRIP
Keyword(s):  
Structural Changes ◽  
Expression System ◽  
Point Mutations ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Heterogeneous Distribution ◽  
Functional Consequences ◽  
Transfer Signal ◽  
Translational Process

Expression in vitro with the recombinant baculovirus expression system showed correct biosynthesis and post-translational processing of ‘wild-type’ bovine opsin with regard to translocation, glycosylation, palmitoylation and targeting. However, several of these processes were severely affected by point mutations. From the overall results of 16 mutants reported here, four groups were distinguished. One group significantly affected neither biosynthesis nor folding of opsin (D83N, P291A, A299C-V300A-P303G). A second group produced a truncated protein (R69H, Y301F), suggesting that these positions are essential for a correct translational process. A third group affected membrane translocation as well as glycosylation, which can be interpreted as interference with the function of a transfer signal. Substitutions at positions Glu-113, Glu-122, Glu-134, Arg-135 and Lys-248 belong to this category. A fourth group induced structural changes in the protein that led to heterogeneous distribution in the plasma membrane (E113Q/D, W265F, Y268S). Taking any functional consequences of these mutations into consideration, it seems that point mutations can have mosaic effects and therefore should be examined at several levels (folding, targeting, functional parameters).

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 Heterologous Expression and Purification Systems for Structural Proteomics of Mammalian Membrane Proteins

2002 ◽  
Vol 3 (6) ◽  
pp. 511-517 ◽  
Author(s):  
Isabelle Mus-Veteau
Keyword(s):  
Membrane Proteins ◽  
Heterologous Expression ◽  
Large Scale ◽  
Structural Information ◽  
Structural Data ◽  
Structural Proteomics ◽  
Scale Production ◽  
Expression And Purification ◽  
Large Scale Production ◽  
Heterologous Expression Systems

Membrane proteins (MPs) are responsible for the interface between the exterior and the interior of the cell. These proteins are implicated in numerous diseases, such as cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension and Alzheimer's disease. However, studies on these disorders are hampered by a lack of structural information about the proteins involved. Structural analysis requires large quantities of pure and active proteins. The majority of medically and pharmaceutically relevant MPs are present in tissues at very low concentration, which makes heterologous expression in large-scale production-adapted cells a prerequisite for structural studies. Obtaining mammalian MP structural data depends on the development of methods that allow the production of large quantities of MPs. This review focuses on the different heterologous expression systems, and the purification strategies, used to produce large amounts of pure mammalian MPs for structural proteomics.

Analysis of recombinant tagged equilibrative nucleoside transporter 1 (ENT1) expressed in E. coliThis paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

2011 ◽  
Vol 89 (2) ◽  
pp. 246-255 ◽  
Author(s):  
German Reyes ◽  
Nicole M.I. Nivillac ◽  
Maria Chalsev ◽  
Imogen R. Coe
Keyword(s):  
Membrane Proteins ◽  
Large Scale ◽  
Peer Review Process ◽  
Three Dimensional ◽  
Drug Uptake ◽  
Dimensional Structure ◽  
Parasitic Infections ◽  
Scale Production ◽  
Nucleoside Transporter ◽  
Intracellular Loop

Nucleoside transporters (NTs) are integral membrane proteins necessary for the cellular entry of nucleoside analog drugs used in chemotherapeutic treatment of conditions such as cancer and viral or parasitic infections. NTs are also the targets of certain drugs used in the treatment of various cardiovascular conditions. Because of the importance of NTs in drug uptake, determination of the three-dimensional structure of these proteins, particularly hENT1, has the potential to improve these treatments through structure-based design of more specifically targeted and transported drugs. In this paper, we use NMR spectroscopy to investigate the structure of the large intracellular loop between transmembrane domains 6 and 7 and we also describe a method for the successful overexpression of full-length hENT1 in a bacterial system. Recombinant tandem histidine-affinity (HAT) and 3×FLAG tagged hENT1 was overexpressed in E. coli, affinity purified, and functionally characterized by nitrobenzylthioinosine (NBTI) binding. Anti-3×FLAG immunodetection confirmed the expression of N-HAT-3×FLAG-hENT1, while increased NBTI binding (3.2-fold compared with controls) confirmed the conformational integrity of the recombinant hENT1 within the bacterial inner membrane. Yields of recombinant hENT1 using this approach were ∼15 µg/L of bacterial culture and this approach provides a basis for large-scale production of protein for a variety of purposes.

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