Expression and purification of 15 kDa granulysin utilizing an insect cell secretion system

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Abstract

Granulysin is an antimicrobial and proinflammatory protein expressed in activated human T cells and natural killer cells. A single mRNA produces the 15 kDa isoform which is then cleaved at the amino and carboxy termini to produce the 9 kDa isoform. Recombinant 9 kDa granulysin has been studied in detail but little is known about the function of the 15 kDa isoform, and no protocol has been published describing expression and purification of this form. Two commercially available preparations of the recombinant 15 kDa granulysin contain tags that may affect function. Here we describe for the first time a method to produce 15 kDa granulysin as a secreted protein from insect cells. The 15 kDa granulysin is purified using a HiTrap Heparin column and a Resource S column. A typical a yield of purified 15 kDa granulysin is 0.6 mg/L of insect cell supernatant.

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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