Expression and purification of 15 kDa granulysin utilizing an insect cell secretion system.
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.
Finn
Michael W
MW
Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Room 2016, Bethesda, MD 20892-4256, USA.
Clayberger
Carol
C
Krensky
Alan M
AM
D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte / Q000235:genetics* / Q000302:isolation & purification*; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007313:Insecta / Q000166:cytology*; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification
D000945:Antigens, Differentiation, T-Lymphocyte; C075005:GNLY protein, human; D020033:Protein Isoforms