High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles
| Autor: | Viktor Menart, Ana Lenassi Zupan, Sonja Trobec, Vladka Gaberc-Porekar |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Sarcosine
Gene Expression Bioengineering Applied Microbiology and Biotechnology Pichia Pichia pastoris Green fluorescent protein law.invention chemistry.chemical_compound Confocal microscopy law Microscopy Confocal biology General Medicine biology.organism_classification Protein subcellular localization prediction Fluorescence Recombinant Proteins Alcohol oxidase Microscopy Fluorescence chemistry Biochemistry Cytoplasm Biophysics Artifacts Biotechnology |
| Zdroj: | Journal of Biotechnology. 109:115-122 |
| ISSN: | 0168-1656 |
| DOI: | 10.1016/j.jbiotec.2003.11.013 |
| Popis: | Wild type gene for green fluorescent protein (GFP) was stably integrated into the Pichia pastoris genome and yielded an expression level of over 40% of total cellular protein. The high cytoplasmic concentration of fluorescent (properly folded and processed) GFP caused the formation of fluorescent spherical structures, which could be observed by fluorescence or confocal microscopy after controlled permeabilization of the yeast cells with 0.2% N-lauroyl sarcosine (NLS). Fluorescent GFP particles were also isolated after removal of the cell wall and found to be quite resistant to 0.2% N-lauroyl sarcosine. SDS-PAGE analysis of the isolated fluorescent particles revealed the presence of an 80 kDa protein (alcohol oxidase) and GFP (30%). We conclude that GFP is able to enter spontaneously into the peroxisomes and is inserted into densely packed layers of alcohol oxidase. Consequently, the formation of similar fluorescent particles can also be expected in other organisms when using high-level expression systems. As GFP is widely used in fusion with other proteins as a reporter for protein localization and for many other applications in biotechnology, care must be taken to avoid false interpretations of targeting or trafficking mechanisms inside the cells. In addition, when whole cells or cytoplasmic fractions are used for the quantitative determination of GFP levels, incorrect and misleading values of GFP could be obtained due to the formation of fluorescent particles containing material inside which is not available for fluorescence measurements. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect