Fusion of green fluorescent protein to the C-terminus of granulysin alters its intracellular localization in comparison to the native molecule

Journal of Negative Results in Biomedicine
D A Hanson, Steven F Ziegler

Abstract

The engineering of green fluorescent protein (GFP) fusion constructs in order to visibly tag a protein of interest has become a commonly used cell biology technique. Although caveats to this approach are obvious, literature reports in which the chimeric molecule behaves differently than the native molecule are scant. This brief report describes one such case. Granulysin, a small lytic and antimicrobial protein produced by cytotoxic lymphocytes, traffics to the regulated secretory system and is subsequently released from cells upon proper stimulus. In an attempt to elucidate mechanisms by which it accumulates in and is released from cytolytic granules, GFP was fused to the C-terminus of granulysin and expressed in an NK cell line. A control construct expressing the native protein was similarly expressed. The data demonstrate that, while the fusion protein is expressed and secreted, its subcellular localization is altered in comparison to native granulysin. Thus, the addition of GFP to the C-terminus of granulysin obscures the signal(s) that cytotoxic lymphocytes use to sort it to the regulated secretory pathway despite its normal biosynthesis and secretion. This example is offered as a cautionary account for other researchers wh...Continue Reading

References

Apr 9, 1998·Biochimica Et Biophysica Acta·L J PageG M Griffiths
Aug 17, 1999·Molecular Immunology·D A HansonA M Krensky
Feb 28, 2002·Annual Review of Immunology·John H Russell, Timothy J Ley

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Citations

Jul 10, 2010·Proceedings of the National Academy of Sciences of the United States of America·Hai-Bo WangXu Zhang
Aug 29, 2007·DNA and Cell Biology·Bahareh Badrian, Marie A Bogoyevitch
Mar 12, 2013·Human Molecular Genetics·Caroline VanceChristopher E Shaw
Jan 26, 2007·Journal of the Royal Society, Interface·Philippe MarguetLingchong You
Sep 13, 2006·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Brendan BinghamJeffrey D Kennedy
Jul 31, 2016·Plant Cell Reports·Yiqiong LiHongbo Gao
Jun 24, 2008·Molecular Biology Reports·Yukiko HoriikeTakeshi Sekiguchi
Sep 26, 2019·ACS Synthetic Biology·Anton Du Preez Van StadenCarine Smith
Oct 27, 2009·Progress in Neurobiology·Xu ZhangGuo-Qiang Ma

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Methods Mentioned

BETA
transfectant
confocal microscopy

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