Strains, media, and DNA
Escherichia coli
bacterial strain XL10-Gold (Stratagene, USA) was used as a host for
plasmid constructions and was cultured in Luria–Bertani broth (1%
tryptone, 0.5% yeast extract, and 1% sodium chloride) supplemented with
ampicillin (50 μg/ml) or zeocin (25 μg/ml) when appropriate. The P. pastoris yeast strain
X-33 and SMD1168H (Invitrogen, Carlsbad, CA) was cultured on YPD (1%
yeast extract, 2% peptone, and 2% glucose) supplemented with zeocin
(100 μg/ml) when appropriate. YPD with zeocin was also used when
plating yeast that was transformed by electroporation. For solid media,
1.5% agar was used for both bacterial and yeast platings. All media
components were manufactured by Difco Co. (USA), and all enzymes were
purchased from Takara (Japan) unless otherwise noted. Pichia
expression vectors including pGAPZB and pPICZα were purchased from
Invitrogen. Restriction enzyme digestions, plasmid engineering, and
standard techniques were performed as specified elsewhere (Sambrook et
al. 2001). Escherichia coli transformation was performed by the CaCl2
method, and yeast was transformed by electroporation with the Mini
Gene-Pulser apparatus (Biorad) as specified by the manufacturer. Micrococcus luteus IAM1053 (KCCM11576T) was obtained from the Korean Culture Center of Microorganisms (Seoul, Korea).
PCR amplification and cloning of the human LL-37 coding sequences
Two
PCR primers (Primer F1: 5′-ATG AAG ACC CAA AGG GAT GG-3′, Primer R1:
5′-CTA GGA CTC TGT CCT GGG TA-3′) spanning 513 bp of the full-length
human cathelicidin cDNA coding region [Genbank Accession No. NM004345]
were synthesized and used for PCR cloning. The touchdown PCR condition
was 40 cycles of 10 s at 98°C, 1 min at 62°C/50°C, 1 min at 72°C after
denaturing for 5 min at 94°C. The 111 bp of LL-37 gene coding for
antimicrobial peptide at C-terminal end was amplified from nested-PCR
using internal primers (Primer F2: 5′-CCC GAA TTC ACC ATG CTG CTG GGT GAT TTC TTC C-3′, R2; 5′-GCG GCC GCC TAG GAC TCT GTC CTG GGT A-3′), which contain internal EcoRI and NotI enzyme sites, respectively. PCR product was ligated into the pGEM T-Easy vector (Promega, USA) and its DNA sequence was
verified by DNA sequencing.
Construction of the pGAPZ-E/LL-37 expression vector
The strong constitutive GAP promoter (Waterham et al. 1997) was used for expression of the LL-37 gene in P. pastoris. The Pichia-specific
autonomous replication sequence (PARS1) was cloned from PCR using
genomic DNA of X-33 strain as template and inserted into the pGAPZB
vector (Invitrogen, USA) as a replication origin for the episomal state
of the plasmid (Lee et al. 2005).
The LL-37 gene PCR product was ligated into EcoRI/NotI-cleaved pGAPZ-E, which yielded pGAPZ-E/LL37, and it was subsequently transformed into P. pastoris strain X-33. Pichia transformants harboring recombinant pGAPZ-E/LL37 were verified by colony PCR analysis.
Peptide PAGE gel
Pichia
transformants of X-33 were grown at 30°C for 2 days in 20 ml of YPD
broth with Zeocin (100 μg/ml). When their optical density at 600 nm
reached about 30, they were centrifuged and the wet cell weight for
each culture was measured. About 0.5 g of wet cells were resuspended in
0.5 ml PBS buffer with protease inhibitor cocktail (Sigma). The cell
suspension was transferred into a 2 ml mini bead-beater tube; 0.5 g
acid-washed glass beads (425–600 diam., Sigma) were added to the cell
suspension, and cell breakage was done with a Biospec Mini-Beadbeater
(3 × 30 s treatments at 5,000 rpm with 1 min interval cooling on ice).
The lysate was centrifuged for 10 min at 6,000×g and then the supernatant was further centrifuged for 15 min at 12,000×g. About 2∼3 μl of clear supernatant was subjected to 17% PeptiGelTM peptide PAGE analysis (Elpisbio, Korea) using a Bio-rad Mini II kit.
NanoLC-ESI-MS/MS analysis for protein identification
The approximately 5 kDa band detected on SDS-PAGE was manually excised from the gel. The gel pieces were subsequently destained
with destainer solution (100 mM sodium thiosulfate/30 mM potassium ferricyanate), washed with milli-Q water, and rehydrated with 200 mM
ammonium bicarbonate. The pieces were then washed several times with
milli-Q water for 15 min each time, dehydrated with acetonitrile for
5 min, dried, and rehydrated with a total of 200∼300 ng of sequencing
grade, modified trypsin (Promega, Madison, WI, USA) in 25 mM
ammonium bicarbonate, pH 8.5, at 37°C for 16 h. Following digestion,
tryptic peptides were extracted twice with 50% (v/v) acetonitrile
containing 0.5% trifluoroacetic acid for 15 min each time with moderate
vortexing. The extracted solutions were pooled and evaporated to
dryness under vacuum. For ESI-MS/MS analysis, dry peptide samples were
redissolved in 0.1% TFA and purified by C18 Zip-Tip chromatography
(Millipore, Billerica, MA, USA) according to the manufacturer’s
instructions.
Direct 1-D LC-nanoESI-MS/MS
analyses were performed on an integrated nanoLC-MS/MS system (Waters
Co., Japan) comprising a three-pumping Micromass/Waters CapLC system
with an autosampler, a stream select module configured for precolumn
plus analytical capillary column, and a Micromass Q-Tof Global mass
spectrometer fitted with nano-LC sprayer, operated under MassLynx 4.0
control (Micromass, Manchester, UK).
Antimicrobial analysis of recombinant LL-37
Antimicrobial
activity was measured by determining the clear zone around a
cylindrical stainless steel tube (6 mm inner diam. × 10 mm h) against
bacterial lawn of test strain M. luteus IAM1056. Autoclaved NB broth with 1.5% agar was cooled down up to 45°C and poured into 9 cm diam Petri dish with 0.1% of
overnight-grown M. luteus strain. After it was solidified, sterile cylindrical tubes filled with 200 μl of cleared lysate from Pichia transformants were put onto bacterial plates. The plate was incubated at 30°C for 2 days and its clear zone was determined.
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