scholarly journals Integrated Recombinant Protein Expression and Purification Platform Based on Ralstonia eutropha

2005 ◽  
Vol 71 (10) ◽  
pp. 5735-5742 ◽  
Author(s):  
Gavin C. Barnard ◽  
Jesse D. McCool ◽  
David W. Wood ◽  
Tillman U. Gerngross
Keyword(s):  
Protein Purification ◽  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Recombinant Protein Expression ◽  
Specific Interaction ◽  
Single Step ◽  
Protein Expression And Purification ◽  
Model Protein ◽  
Purification Methods ◽  
Green Fluorescent

ABSTRACT Protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. We describe a protein purification scheme wherein Ralstonia eutropha is used to produce its own “affinity matrix,” thereby eliminating the need for external chromatographic purification steps. This approach is based on the specific interaction of phasin proteins with granules of the intracellular polymer polyhydroxybutyrate (PHB). By creating in-frame fusions of phasins and green fluorescent protein (GFP) as a model protein, we demonstrated that GFP can be efficiently sequestered to the surface of PHB granules. In a second step, we generated a phasin-intein-GFP fusion, wherein the self-cleaving intein can be activated by the addition of thiols. This construct allowed for the controlled binding and release of essentially pure GFP in a single separation step. Finally, pure, active β-galactosidase was obtained in a single step using the above described method.

scholarly journals Effects of Recombinant Protein Expression on Green Fluorescent Protein Diffusion inEscherichia coli

Biochemistry ◽  
2009 ◽  
Vol 48 (23) ◽  
pp. 5083-5089 ◽  
Author(s):  
Kristin M. Slade ◽  
Rachael Baker ◽  
Michael Chua ◽  
Nancy L. Thompson ◽  
Gary J. Pielak
Keyword(s):  
Green Fluorescent Protein ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Fluorescent Protein ◽  
Recombinant Protein Expression ◽  
Inescherichia Coli ◽  
Protein Diffusion ◽  
Green Fluorescent

scholarly journals A positive Selection Escherichia Coli Recombinant Protein Expression Vector for One-Step Cloning

2022 ◽  
Vol 9 ◽  
Author(s):  
Shinto James ◽  
Vikas Jain
Keyword(s):  
Recombinant Protein ◽  
Fluorescent Protein ◽  
Recombinant Protein Expression ◽  
Plasmid Vector ◽  
Cost Effective ◽  
Single Step ◽  
Selection Strategy ◽  
Host Bacterium ◽  
T7 Promoter ◽  
Vector Systems

We introduce OLIVAR (Orientation seLection of Insert in Vector through Antisense Reporter) as a novel selection strategy for the insertion of protein-coding genes into vector backbones. As a proof-of-concept, we have engineered a plasmid vector, pGRASS (Green fluorescent protein Reporter from Antisense promoter-based Screening System), for gene cloning in E. coli. With pGRASS, positive clones can be effortlessly distinguished from negative clones after blunt-end cloning. The vector not only screens clones with an insert but also for its correct orientation. The design further allows for the expression of recombinant protein from the T7 promoter in an appropriate host bacterium. With this vector, we are able to reduce the entire cloning workflow into a single step involving a 2-h reaction at room temperature. We believe that our cloning-cum-screening system presented here is extremely cost-effective and straightforward and can be applied to other vector systems and domains such as phage display and library construction.

scholarly journals Roles of NUDE and NUDF Proteins of Aspergillus nidulans: Insights from Intracellular Localization and Overexpression Effects

2003 ◽  
Vol 14 (3) ◽  
pp. 871-888 ◽  
Author(s):  
Vladimir P. Efimov
Keyword(s):  
Green Fluorescent Protein ◽  
Aspergillus Nidulans ◽  
Heavy Chain ◽  
Fluorescent Protein ◽  
Specific Interaction ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Dynein Heavy Chain ◽  
Terminal Domain ◽  
Green Fluorescent

The NUDF protein of the filamentous fungus Aspergillus nidulans functions in the cytoplasmic dynein pathway. It binds several proteins, including the NUDE protein. Green fluorescent protein-tagged NUDF and NUDA (dynein heavy chain) localize to linearly moving dashes (“comets”) that coincide with microtubule ends. Herein, deletion of the nudE gene did not eliminate the comets of NUDF and NUDA, but affected the behavior of NUDA. Comets were also observed with the green fluorescent protein-tagged NUDE and its nonfunctional C-terminal domain. In addition, overexpressed NUDA and NUDE accumulated in specks that were either immobile or bounced randomly. Neither comets nor specks were observed with the functional N-terminal domain of NUDE, indicating that these structures are not essential for NUDE function. Furthermore, NUDF overproduction totally suppressed deletion of the nudEgene. This implies that the function of NUDE is secondary to that of NUDF. Unexpectedly, NUDF overproduction inhibited one conditionalnudA mutant and all tested apsA mutants. An allele-specific interaction between the nudF andnudA genes is consistent with a direct interaction between NUDF and dynein heavy chain. Because APSA and its yeast homolog Num1p are cortical proteins, an interaction between thenudF and apsA genes suggests a role for NUDF at the cell cortex.

scholarly journals The Ralstonia eutropha H16 phasin PhaP1 is targeted to intracellular triacylglycerol inclusions in Rhodococcus opacus PD630 and Mycobacterium smegmatis mc2155, and provides an anchor to target other proteins

Microbiology ◽  
2006 ◽  
Vol 152 (11) ◽  
pp. 3271-3280 ◽  
Author(s):  
Jan Hänisch ◽  
Marc Wältermann ◽  
Horst Robenek ◽  
Alexander Steinbüchel
Keyword(s):  
Fusion Protein ◽  
Mycobacterium Smegmatis ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Rhodococcus Opacus ◽  
Egfp Fusion Protein ◽  
Green Fluorescent ◽  
Cell Fractions

In Ralstonia eutropha, the H16 phasin PhaP1 represents the major phasin that binds to the surface of polyhydroxyalkanoate (PHA) inclusions. In this study, C-terminal fusions of PhaP1 with enhanced green fluorescent protein (eGFP) and with Escherichia coli β-galactosidase (LacZ) were expressed separately in the triacylglycerol (TAG)-accumulating actinomycetes Rhodococcus opacus PD630 and Mycobacterium smegmatis mc2155, employing the M. smegmatis acetamidase (ace) promoter of the Escherichia–Mycobacterium/Rhodococcus shuttle plasmid pJAM2. PhaP1 and the PhaP1 fusion proteins were expressed stably in the recombinant strains. Western blot analysis of cell fractions of Rh. opacus revealed that PhaP1 and the PhaP1–eGFP fusion protein were associated with the TAG inclusions, whereas no phasin or phasin fusion protein was detected in the soluble and membrane fractions. Additional electron microscopy/immunocytochemistry studies demonstrated that PhaP1 was mainly located on the surface of intracellular TAG inclusions; in addition, some PhaP1 also occurred at the plasma membrane. Fluorescence microscopic investigations of the subcellular distribution of the PhaP1–eGFP fusion protein in vivo and on isolated TAG inclusions revealed that the fusion protein was bound to TAG inclusions at all stages of their formation, and to some extent at the cytoplasmic membrane. The PhaP1–LacZ fusion protein also bound to the TAG inclusions, and could be separated together with the inclusions from Rh. opacus crude extracts, thus demonstrating the immobilization of β-galactosidase activity on the inclusions. This is believed to be the first report demonstrating the ability of PhaP1 to bind to lipid inclusions in addition to PHA inclusions. Furthermore, it was demonstrated that this non-specificity of PhaP1 can be utilized to anchor enzymically active fusion proteins to a matrix of bacterial TAG inclusions.

scholarly journals Human Cytomegalovirus Labeled with Green Fluorescent Protein for Live Analysis of Intracellular Particle Movements

2005 ◽  
Vol 79 (5) ◽  
pp. 2754-2767 ◽  
Author(s):  
Kerstin Laib Sampaio ◽  
Yolaine Cavignac ◽  
York-Dieter Stierhof ◽  
Christian Sinzger
Keyword(s):  
Green Fluorescent Protein ◽  
Human Cytomegalovirus ◽  
Fluorescent Protein ◽  
Rare Event ◽  
Single Step ◽  
Wild Type Virus ◽  
Cell Cortex ◽  
Long Distance ◽  
Infected Cells ◽  
Green Fluorescent

ABSTRACT Human cytomegalovirus (HCMV) replicates in the nuclei of infected cells. Successful replication therefore depends on particle movements between the cell cortex and nucleus during entry and egress. To visualize HCMV particles in living cells, we have generated a recombinant HCMV expressing enhanced green fluorescent protein (EGFP) fused to the C terminus of the capsid-associated tegument protein pUL32 (pp150). The resulting UL32-EGFP-HCMV was analyzed by immunofluorescence, electron microscopy, immunoblotting, confocal microscopy, and time-lapse microscopy to evaluate the growth properties of this virus and the dynamics of particle movements. UL32-EGFP-HCMV replicated similarly to wild-type virus in fibroblast cultures. Green fluorescent virus particles were released from infected cells. The fluorescence stayed associated with particles during viral entry, and fluorescent progeny particles appeared in the nucleus at 44 h after infection. Surprisingly, strict colocalization of pUL32 and the major capsid protein pUL86 within nuclear inclusions indicated that incorporation of pUL32 into nascent HCMV particles occurred simultaneously with or immediately after assembly of the capsid. A slow transport of nuclear particles towards the nuclear margin was demonstrated. Within the cytoplasm, most particles performed irregular short-distance movements, while a smaller fraction of particles performed centripetal and centrifugal long-distance movements. Although numerous particles accumulated in the cytoplasm, release of particles from infected cells was a rare event, consistent with a release rate of about 1 infectious unit per h per cell in HCMV-infected fibroblasts as calculated from single-step growth curves. UL32-EGFP-HCMV will be useful for further investigations into the entry, maturation, and release of this virus.

Mild and cost-effective green fluorescent protein purification employing small synthetic ligands

2015 ◽  
Vol 1418 ◽  
pp. 83-93 ◽  
Author(s):  
Ana Sofia Pina ◽  
Ana Margarida G.C. Dias ◽  
Fatma Isik Ustok ◽  
Graziella El Khoury ◽  
Cláudia S.M. Fernandes ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Purification ◽  
Fluorescent Protein ◽  
Cost Effective ◽  
Green Fluorescent

scholarly journals Green fluorescent protein purification through Immobilized Metal Affinity Chromatografy (IMAC) and its relevance for Biomedical Science students during Biochemistry practical classes at La Trobe University – Australia

2016 ◽  
Vol 14 (3) ◽  
pp. 34
Author(s):  
Alex Jose José De Melo Silva ◽  
Lumar Lucena Alves ◽  
Julian Pakay
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Purification ◽  
Fluorescent Protein ◽  
Biomedical Science ◽  
Science Students ◽  
Metal Affinity ◽  
Green Fluorescent

Este trabalho foi realizado como prática integrada da disciplina de Bioquímica no curso de graduação em Ciências Biomédicas, objetivando demonstrar técnicas utilizadas para a purificação da Proteina Fluorescente Verde (GFP).  Para realização dos experimentos foi utilizada como metodologia principal a Cromatografia de Afinidade por Metal Imobilizado (IMAC). A região de abertura para alongamento da GFP foi subclonada dentro de um vetor de expressão chamado pQE30. A produção subsequente da proteína marcada com hexahistidina na região N-terminal, facilitou sua purificação pela IMAC. Uma purificação de aproximadamente três vezes mais que a esperada da GFP foi obtida. Dessa forma, os estudantes completaram o curso adquirindo significante experiência em relação às técnicas fundamentais na área molecular relacionadas à clonagem e princípios de expressão e purificação de proteínas recombinantes.

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