In VivoSelf-Assembly of Stable Green Fluorescent Protein Fusion Particles and Their Uses in Enzyme Immobilization

ABSTRACTBacterial inclusion bodies are aggregations of mostly inactive and misfolded proteins. However, previously thein vivoself-assembly of green fluorescent protein (GFP) fusions into fluorescent particles which displayed specific binding sites suitable for applications in bioseparation and diagnostics was demonstrated. Here, the suitability of GFP particles for enzyme immobilization was assessed. The enzymes tested were a thermostable α-amylase fromBacillus licheniformis,N-acetyl-d-neuraminic acid aldolase (NanA) fromEscherichia coli, and organophosphohydrolase (OpdA) fromAgrobacterium radiobacter. Respective GFP particles were isolated and could be stably maintained outside the cell. These enzyme-bearing GFP particles exhibited considerable stability across a range of temperature, pH, and storage conditions and could be recycled. The α-amylase-bearing particles retained activity after treatments at 4 to 85°C and at pHs 4 to 10, were stable for 3 months at 4°C, and could be recycled up to three times. OpdA-bearing particles retained degradation activity after treatments at 4 to 45°C and at pHs 5 to 10 and were able to be recycled up to four times. In contrast, the performance of NanA-bearing particles rapidly declined (>50% loss) after each recycling step and 3 months storage at 4°C. However, they were still able to convertN-acetylmannosamine and pyruvate toN-acetylneuraminic acid after treatment at 4 to 85°C and at pHs 4 to 11. Fluorescent GFP fusion particles represent a novel method for the immobilization and display of enzymes. Potential applications include diagnostic assays, biomass conversion, pharmaceutical production, and bioremediation.

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The Sudden Recruitment of γ-Tubulin to the Centrosome at the Onset of Mitosis and Its Dynamic Exchange Throughout the Cell Cycle, Do Not Require Microtubules

The Journal of Cell Biology ◽

10.1083/jcb.146.3.585 ◽

1999 ◽

Vol 146 (3) ◽

pp. 585-596 ◽

Cited By ~ 238

Author(s):

Alexey Khodjakov ◽

Conly L. Rieder

Keyword(s):

Cell Cycle ◽

Green Fluorescent Protein ◽

Fluorescence Recovery After Photobleaching ◽

Fluorescent Protein ◽

Green Fluorescent Protein Fusion ◽

Protein Fusion ◽

Green Fluorescent ◽

Dynamic Exchange ◽

Two Populations

γ-Tubulin is a centrosomal component involved in microtubule nucleation. To determine how this molecule behaves during the cell cycle, we have established several vertebrate somatic cell lines that constitutively express a γ-tubulin/green fluorescent protein fusion protein. Near simultaneous fluorescence and DIC light microscopy reveals that the amount of γ-tubulin associated with the centrosome remains relatively constant throughout interphase, suddenly increases during prophase, and then decreases to interphase levels as the cell exits mitosis. This mitosis-specific recruitment of γ-tubulin does not require microtubules. Fluorescence recovery after photobleaching (FRAP) studies reveal that the centrosome possesses two populations of γ-tubulin: one that turns over rapidly and another that is more tightly bound. The dynamic exchange of centrosome-associated γ-tubulin occurs throughout the cell cycle, including mitosis, and it does not require microtubules. These data are the first to characterize the dynamics of centrosome-associated γ-tubulin in vertebrate cells in vivo and to demonstrate the microtubule-independent nature of these dynamics. They reveal that the additional γ-tubulin required for spindle formation does not accumulate progressively at the centrosome during interphase. Rather, at the onset of mitosis, the centrosome suddenly gains the ability to bind greater than three times the amount of γ-tubulin than during interphase.

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In vivo monitoring of intracellular expression of human interleukin-2 using green fluorescent protein fusion partner in Pichia pastoris

Biotechnology Letters ◽

10.1023/b:bile.0000035489.09417.d4 ◽

2004 ◽

Vol 26 (14) ◽

pp. 1157-1162 ◽

Cited By ~ 11

Author(s):

Hyung Joon Cha ◽

Nimish G. Dalal ◽

William E. Bentley

Keyword(s):

Green Fluorescent Protein ◽

Pichia Pastoris ◽

Fluorescent Protein ◽

Interleukin 2 ◽

Fusion Partner ◽

Green Fluorescent Protein Fusion ◽

Protein Fusion ◽

Intracellular Expression ◽

Green Fluorescent

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Weak Transcription of thecry1AcGene in Nonsporulating Bacillus thuringiensis Cells

Applied and Environmental Microbiology ◽

10.1128/aem.01229-12 ◽

2012 ◽

Vol 78 (18) ◽

pp. 6466-6474 ◽

Cited By ~ 17

Author(s):

Hui Yang ◽

Pinshu Wang ◽

Qi Peng ◽

Rong Rong ◽

Chunxia Liu ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Bacillus Thuringiensis ◽

Fluorescent Protein ◽

Cellular Level ◽

Protein Fusion ◽

Reporter System ◽

Content Type ◽

Reverse Transcription Pcr ◽

Green Fluorescent ◽

Sigma E

ABSTRACTThecry1Acgene ofBacillus thuringiensissubsp.kurstakiHD-73 (B. thuringiensisHD-73) is a typical example of a sporulation-dependent crystal gene and is controlled by sigma E and sigma K during sporulation. To monitor the production and accumulation of Cry1Ac at the cellular level, we developed a green fluorescent protein-based reporter system. The production of Cry1Ac was monitored inspo0A,sigE, andsigKmutants, and these mutants were able to express the Cry1Ac-green fluorescent protein fusion protein. In nonsporulatingB. thuringiensisHD-73 cells, low-level expression ofcry1Acwas also observed. Reverse transcription-PCR and Western blotting results confirmed that thecry1Acpromoter has low activity in nonsporulatingB. thuringiensiscells. A beta-galactosidase assay demonstrated that the transcription of thecry1Acgene during exponential and transition phases is positively regulated by Spo0A. Additional bioassay results indicated thatspo0AandsigEmutants containing thecry1Ac-gfpfusion exhibited insecticidal activity againstPlutella xylostellalarvae.

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Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

Biochemical Journal ◽

10.1042/bj3390299 ◽

1999 ◽

Vol 339 (2) ◽

pp. 299-307 ◽

Cited By ~ 33

Author(s):

Arthur L. KRUCKEBERG ◽

Ling YE ◽

Jan A. BERDEN ◽

Karel van DAM

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Green Fluorescent Protein ◽

Glucose Transport ◽

Cellular Localization ◽

Fluorescent Protein ◽

Hexose Transporter ◽

High Concentrations ◽

Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

Cell Transplantation ◽

10.1177/0963689720978219 ◽

2021 ◽

Vol 30 ◽

pp. 096368972097821

Author(s):

Andrea Tenorio-Mina ◽

Daniel Cortés ◽

Joel Esquivel-Estudillo ◽

Adolfo López-Ornelas ◽

Alejandro Cabrera-Wrooman ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Ectopic Expression ◽

Neural Induction ◽

Human Keratinocytes ◽

Differentiation Potential ◽

Neuronal Proteins ◽

Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

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