High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles

2004 ◽  
Vol 109 (1-2) ◽  
pp. 115-122 ◽  
Author(s):  
A Lenassi Zupan
Keyword(s):  
Green Fluorescent Protein ◽  
Pichia Pastoris ◽  
Fluorescent Protein ◽  
High Expression ◽  
Fluorescent Particles ◽  
Green Fluorescent

scholarly journals Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe

2004 ◽  
Vol 70 (2) ◽  
pp. 961-966 ◽  
Author(s):  
Antje Eiden-Plach ◽  
Tatjana Zagorc ◽  
Tanja Heintel ◽  
Yvonne Carius ◽  
Frank Breinig ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Green Fluorescent Protein ◽  
Pichia Pastoris ◽  
Schizosaccharomyces Pombe ◽  
Protein Secretion ◽  
Candida Glabrata ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Foreign Protein ◽  
Green Fluorescent

ABSTRACT Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.

Development of Pichia pastoris as a rumen escape vehicle for the intestinal delivery of recombinant proteins in ruminants

2004 ◽  
Vol 84 (4) ◽  
pp. 611-619 ◽  
Author(s):  
C. E. Strauss ◽  
T. A. McAllister ◽  
L. B. Selinger
Keyword(s):  
Small Intestine ◽  
Green Fluorescent Protein ◽  
Pichia Pastoris ◽  
Recombinant Proteins ◽  
Fluorescent Protein ◽  
Intact Cells ◽  
Ruminal Fluid ◽  
Bioactive Proteins ◽  
Green Fluorescent

The effectiveness of cellular encapsulation as a method for delivery of bioactive proteins and limiting amino acids to the small intestine of ruminants was investigated. Intracellular expression of green fluorescent protein variant (GFPuv) in Pichia pastoris was used as a visible marker to assess the cellular integrity of P. pastoris and determine the potential of this approach for protecting recombinant proteins from microbial proteolysis in the rumen. Fluorescent cells were easily identified in the presence of strained ruminal fluid when viewed by epifluorescent microscopy, and intact cells were readily enumerated. Batch cultures with rumen digesta demonstrated that 93, 97 and 25% of P. pastoris cells remained intact after 36 to 48 h of incubation in clarified ruminal fluid, an isolated bacterial fraction, and whole ruminal fluid, respectively. In continuous culture (Rusitec) with a dilution rate of 0.75 d-1, 19% of P. pastoris cells flowed intact from the fermentation vessels. In vitro abomasal simulations demonstrated that 84% of inoculated P. pastoris had lysed within 12 h of incubation, a property that is necessary for the release of encapsulated protein prior to the small intestine. These in vitro studies suggest that P. pastoris may be an effective vehicle for post-ruminal delivery of bioactive proteins in ruminants. Key words: Ruminal escape vehicle, Pichia pastoris, green fluorescent protein, bypass protein

scholarly journals Peroxisome Degradation by Microautophagy in Pichia pastoris: Identification of Specific Steps and Morphological Intermediates

1998 ◽  
Vol 141 (3) ◽  
pp. 625-636 ◽  
Author(s):  
Yasuyoshi Sakai ◽  
Antonius Koller ◽  
Linda K. Rangell ◽  
Gilbert A. Keller ◽  
Suresh Subramani
Keyword(s):  
Green Fluorescent Protein ◽  
Pichia Pastoris ◽  
Fluorescent Protein ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Complementation Groups ◽  
Green Fluorescent ◽  
Kinetics Of ◽  
Pyridinium Dibromide ◽  
Shed Light

We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy. Structures corresponding to the intermediates were also identified by electron microscopy. The kinetics of appearance and disappearance of these intermediates is consistent with a precursor–product relationship between intermediates, which form the basis of a model for microautophagy. Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version [GFP])-SKL via both autophagic pathways. P. pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprising 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis.

scholarly journals Visualization of Intracellular Movement of Vaccinia Virus Virions Containing a Green Fluorescent Protein-B5R Membrane Protein Chimera

2001 ◽  
Vol 75 (10) ◽  
pp. 4802-4813 ◽  
Author(s):  
Brian M. Ward ◽  
Bernard Moss
Keyword(s):  
Green Fluorescent Protein ◽  
Vaccinia Virus ◽  
Intracellular Transport ◽  
Fluorescent Protein ◽  
Digital Camera ◽  
Immunogold Electron Microscopy ◽  
Intracellular Movement ◽  
Infected Cells ◽  
Fluorescent Particles ◽  
Green Fluorescent

ABSTRACT We produced an infectious vaccinia virus that expressed the B5R envelope glycoprotein fused to the enhanced green fluorescent protein (GFP), allowing us to visualize intracellular virus movement in real time. Previous transfection studies indicated that fusion of GFP to the C-terminal cytoplasmic domain of B5R did not interfere with Golgi localization of the viral protein. To determine whether B5R-GFP was fully functional, we started with a B5R deletion mutant that made small plaques and inserted the B5R-GFP gene into the original B5R locus. The recombinant virus made normal-sized plaques and acquired the ability to form actin tails, indicating reversal of the mutant phenotype. Moreover, immunogold electron microscopy revealed that both intracellular enveloped virions (IEV) and extracellular enveloped virions contained B5R-GFP. By confocal microscopy of live infected cells, we visualized individual fluorescent particles, corresponding to IEV in size and shape, moving from a juxtanuclear location to the periphery of the cell, where they usually collected prior to association with actin tails. The fluorescent particles could be seen emanating from cells at the tips of microvilli. Using a digital camera attached to an inverted fluorescence microscope, we acquired images at 1 frame/s. At this resolution, IEV movement appeared saltatory; in some frames there was no net movement, whereas in others movement exceeded 2 μm/s. Further studies indicated that IEV movement was reversibly arrested by the microtubule-depolymerizing drug nocodazole. This result, together with the direction, speed, and saltatory motion of IEV, was consistent with a role for microtubules in intracellular transport of IEV.

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