lyophilization of NK
The final fermentation broth was collected and centrifuged (GL-25M,
Luxiangyi, Shanghai, China) immediately at 18,300 × g for 10 min. Solid
(NH4)2SO4 was then
gradually added until the supernatant was 20% saturated, after which it
was stored at 4°C overnight. Next, the broth was centrifuged at 18,300 ×
g for 10 min and the precipitate was discarded. Additional
(NH4)2SO4 was
subsequently added to make the supernatant 60% saturated, after which
the mixture was stored at 4°C overnight again. Following centrifugation
at
18,300 × g for 30 min, the supernatant was removed and the precipitate
was dissolved in 10 mmol L-1 PBS (pH 7.4) as a crude
enzyme. The crude enzyme was further purified by Ni–NTA affinity
chromatography according to the manufacturer’s (Novagen, San Diego, CA,
USA) protocols. After purification, 10 µl of eluted fractions of
different dilution times were used to assay their fibrinolytic
activities. Residual solution was desalted by dialysis and lyophilized
using a GLZY-0.5B
vacuum
freeze
drier
(Pudong Freeze Drying Equipment, Shanghai, China) to obtain NK
Lyophilized powder.
SDS-PAGE and Western blotting
analysis
SDS-PAGE protein analysis was performed in a Mini-Protean Tetra system
(BioRad, Hercules, CA, USA) by loading 30 μL of 1: 1 (v: v) boiled
supernatant and dye buffer onto a
4–20%
precast Mini protean TGX gel (BioRad)
and then running the samples in 1 × Tris-glycine-SDS running buffer
(BioRad, Shanghai, China) at 100 V for 15 min followed by 200 V for 25
min. A protein standard (10-250 kDa, Precision Plus Protein
Kaleidoscope, Bio-Rad) was used as a ladder.
NK was identified by Western
blotting analysis as described by Towbin, with slight modification
[18]. Briefly, 10 μL aliquots of different supernatants were
separated by 15% SDS–PAGE. After electrophoresis, the protein bands
were transferred from the gel to an Amersham nitrocellulose (NC)
membrane (GE Healthcare, Piscataway, NJ, USA) using a Mini Trans Blot
electrophoretic transfer cell (BioRad). NK on the membrane was reacted
with a mouse monoclonal antibody (Yisheng Biological Technology,
Shanghai, China) against His-tag (1:1000 v/v), then incubated with
anti-mouse Ig G alkaline phosphatase (Sigma) conjugate (1:2000
v/v). After washing, the NC membrane was stained using Super ECL
Detection Reagent (Yisheng Biology Technology, Shanghai, China)
according to the manufacturer’s
instructions, then covered by an X-ray film and exposed for 1 min. A
Tanon v.3500 Gel Imaging System (Tanon Co., Shanghai, China) was used
for the analysis of proteins.