Expression and purification of 15 kDa granulysin utilizing an insect cell secretion system
Introduction
Granulysin is an antimicrobial and proinflammatory protein expressed in human natural killer and activated T cells [1], [2], [3]. The 15 kDa protein is cleaved at the amino and carboxy termini to produce a 9 kDa isoform [3], [4], [5]. The 9 kDa granulysin lyses gram positive and gram negative bacteria, parasites, fungi, yeast and a variety of tumor cell lines [6], [7], [8]. The 9 kDa granulysin is sequestered in cytolytic granules while the 15 kDa isoform is constitutively secreted from cells [9], [10].
Until recently the study of 15 kDa granulysin has been hampered by the inability to express and purify it. Commercially available recombinant 15 kDa granulysin from R&D Systems and Novus Biologicals both contain tags that may affect function. The 15 kDa granulysin from R&D Systems contains a 10-histidine tag at the C-terminus while the recombinant molecule from Novus Biologicals includes an intact GST tag at the N-terminus. The 15 kDa granulysin made by our protocol has no tag and is purified from insect cell supernatants. We show here that the intact untagged molecule behaves differently from the commercially available tagged forms in a monocyte activation bioassay, suggesting that further understanding the physiologic role of 15 kDa granulysin will be advanced by using the intact, untagged form. This work represents the starting point for structural studies as well as further functional characterization of this isoform of granulysin.
Section snippets
Commercially available 15 kDa granulysin
The 15 kDa granulysin was purchased from R&D Systems (Cat # 3138-GN/CF) and Novus Biologicals (Cat # H00010578-P01).
Construction of 15 kDa granulysin baculovirus expression vector
A cDNA clone of 15 kDa granulysin was generated from human peripheral blood lymphocytes and cloned into pet28A Escherichia coli expression vector. This previously unpublished pet28A construct containing the 15 kDa granulysin was used as the template to generate an insect cell secretion expression system. The first subcloning from pet28A was to Gateway Donor vector pDonr253 which was
Results
A cDNA clone of the 15 kDa granulysin was isolated from human peripheral blood lymphocytes and cloned into the pet28A E. coli expression vector. Several expression studies were carried out using E. coli but poor yields due to protein degradation indicated the pet28A expression system was not feasible (data not shown). The 15 kDa gene was subcloned into an E. coli expression vector containing a GST-tag but instability of the protein and degradation of the GST-tag resulted in poor yields of protein
Discussion
Our group first identified granulysin as a gene expressed by human T lymphocytes “late” (3–5 days) after activation [12], [13], [14]. We expressed the 9 kDa isoform in E. coli and have published more than 45 papers describing its function in cytotoxicity and inflammation [4], [5]. However, we were unable to express the 15 kDa isoform to study its function.
Recently, publications have suggested function(s) for 15 kDa granulysin, using commercially available forms of the protein which retain their
Acknowledgments
This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, and the Center for Cancer Research. We also thank the members of the Protein Expression Laboratory at NCI Frederick.
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