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 (data not shown). Attempts to express 15 kDa granulysin using the yeast Kluyveromyces lactis also were unsuccessful.
The failure of E. coli and K. lactis as viable systems to express 15 kDa granulysin prompted us to explore an insect cell secretion system. The original pet28 clone was used as a template to amplify the 15 kDa granulysin into a modified pDonr201 vector called pDonr253 (see Materials and Methods). The 15 kDa gene was amplified by PCR and a baculovirus GP67 secretion leader sequence was engineered at the 5′ end of granulysin by adapter PCR (see Materials and Methods). The PCR product was purified and recombined into pDonr253 vector according to the manufacturer's protocol. The vector was transformed into E. coli DH5α and plated on LB plates that contained spectinomycin. The plasmid was purified from E. coli and the 15 kDa granulysin sequence was verified.
The verified clone was then subcloned into pDest-670 insect cell expression vector. This expression clone was verified by size and restriction digest pattern. The new expression construct was transformed into E. coli DH10Bac cells and plated on LB plates containing kanamycin, gentamycin, tetracycline, X-gal, and IPTG as per manufacturer's protocols. Only white colonies were selected from the plates and bacmid DNA sequences across the bacmid junctions.
Generation of baculovirus containing 15 kDa granulysin
The purified bacmid DNA described above was transfected into Sf9 insect cells to generate the recombinant baculovirus. Sf9 insect cells were grown to 1.5 × 106 cells/100 ml in SFX medium. Insect Gene Juice was mixed in a ration of 3:1 to bacmid DNA for the transfection into the Sf9 cells. After 4 days the virus was harvested and the baculovirus was then titrated in Sf9 easy titer cells.
Expression of 15 kDa granulysin in Hi5 insect cells
Both Sf9 and Hi5 insect cells were tested for expression of 15 kDa granulysin. In addition to cell type, the effects of time of infection and temperature were also tested (). Based on these studies, we selected Hi5 cells and, after infection, shifted the temperature from 27 °C to 21 °C and allowed the cells to grow for 48 hours.
Figure 1 Western blot of 15 kDa granulysin secreted from insect cells. Different cell types, expression times, and growth temperatures were tested. Each lane contains 88 ug of protein. Lanes 1: Sf9 cells 27 °C for 48 hours, Lane 2: Hi5 cells 21 °C (more ...)
Hi5 insect cells were grown in a disposable 3 liter flask containing 1 liter SFX medium. The cell culture was set to 8.5 × 105/ml and allowed to grow overnight at 27 °C. The next day the cells were counted and then a MOI of 3 was used to infect the cells. The cells were kept at 27 °C for 4 hours to allow the maximum infection, and then the culture was shifted to 21 °C and grown for 48 hours. The cells were then spun down and only the cell supernatant was used for purification.
Purification of 15 kDa granulysin
A purification protocol was optimized using 500 ml of insect cell supernatant (). The material was thawed at 4 °C and filtered through a 0.45 um filter. To initially concentrate the 15 kDa granulysin, we elected to take advantage of the high isoelectric point of 15 kDa granulysin (pI = 9.39). The filtered insect supernatant was loaded onto a 5 ml HiTrap Heparin HP column at a loading speed of 5 ml per minute. The protein was eluted using NaCl and 15 kDa granulysin eluted at a conductivity range between 55-68 mS/cm.
This material was then pooled and buffer exchanged back into Hepes buffered solution containing less then 50 mM NaCl. Again taking advantage of the positive charge of 15 kDa granulysin, the cation exchange column Resource S was used as the final step in the purification of 15 kDa granulysin. The material was then injected onto an equilibrated 1 ml Resource S column. Protein was eluted in the same buffer used in the heparin purification and 15 kDa granulysin eluted over a conductivity range of 48-53mS/cm. At this stage the protein was concentrated and a BCA assay was used to determine the concentration of the protein. The protein was then run on a 15% SDS-PAGE gel to verify purity (). A Western blot and mass spectrometry were also performed on the purified protein to verify that it as the 15 kDa form of granulysin (data not shown).
Figure 2 15% SDS-PAGE stained with coomassie of purification of 15 kDa granulysin secreted from Hi5 insect cells. Lane 1: Standard (250 kDa, 150 kDa, 100 kDa, 75 kDa, 50 kDa, 37 kDa, 25 kDa, 20 kDa, 15 kDA, 10 kDa), Lane 2: 7 ug insect supernatant, Lane 3: 3.5 (more ...)
shows a typical yield from 500 ml of insect cell supernatant. Normally two 500 ml purifications are combined, resulting in 0.6 mg of purified 15 kDa granulysin from 1 liter of material. Aliquots of the purified protein were prepared, snap frozen in liquid nitrogen and stored at -80 °C. The purified protein was tested for LPS using the Limulus Amebocyte Lysate Kinetic −QCL kit by Lonza(see Materials and Methods); all preparations contained <1 EU LPS/ml.
We attempted to scale up the purification protocol by either concentrating the insect supernatant prior to loading onto a column or using larger columns for the purification. A variety of concentration methods failed because the protein either precipitated out of solution or stuck to membranes used to concentrate the protein. In other cases it was quicker to directly load the material onto a column instead of trying to concentrate it under a stream of nitrogen.
The use of larger columns also proved to be problematic. The best yield was obtained using a 5 ml HiTrap Heparin HP and loading 500 ml of insect supernatant at a time. A larger 20 ml heparin column is available but it only comes in a fast flow bead form instead of the high performance type. Simply connecting two or three 5 ml HiTrap columns also resulted in decreased purity of 15 kDa granulysin for reasons we do not understand.
Comparison of 15 kDa granulysin with commercial available forms of the protein
The purified 15 kDa granulysin was then compared to the two commercially available forms of 15 kDa granulysin. The Novus Biologicals form contains an intact GST-Tag while the R&D Systems product has a C-terminal 10-histidine tag. We have previously shown that 15 kDa granulysin activates monocytes, causing changes in gene expression (Clayberger et. al. submitted). We compared the ability of the three forms of granulysin to promote expression of IL-6 and CCL20, as measured by real time PCR. Over a wide range of protein (1-200 nM) our untagged 15 kDa granulysin was superior to either of the commercial preparations ().
Figure 3 Comparison of three different sources of 15 kDa granulysin. Monocytes were cultured for 4 hours with the indicated concentrations of granulysin. Expression of IL-6 and CCL20 was measured by rtPCR and is depicted as fold increase over expression in monocytes (more ...)