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Legume Genomics and Genetics (online), 2011, Vol. 2, No.2, 6-13
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12
reached 0.6, IPTG was added with a final concen-
tration of 0.1mmol/L to induce target fusion protein.
Under the shaking culture at the speed of 200 r/min
at 30
℃
, the recombinant strains were collected after
induced for 0 min, 30 min, 60 min, 120 min, 180 min,
240 min, 300 min and 360 min, respectively. The su-
pernatant was discarded by centrifugation at 13 000 r/min
at 4 for 1 min
℃
, and precipitate was re-suspended
with PBS, then added
equivalent 2×SDS sample
buffer, denatured for 5 min at 100
℃
and cooled on ice
bath for 5 min, finally centrifugated at 13 000 r/min at
4 for 5 min. Took about 20 μL of t
℃
he samples used
for SDS-PAGE electrophoresis.
Protein concentration was measured by the BCA
method (Walker, 1994). 37.5 μL (8 mg/mL) BSA was
added into 262.5 μL ddH
2
O to make protein standard
solution with final BSA concentration 1 mg/mL, took
150
L solution diluted 7 times consecutively to
prepare BSA diluting standard solution with the con-
centrations at 500
g/mL, 250
g/mL, 125
g/mL,
62.5
g/mL, 31.25
g/mL, 15.625
g/mL, and
7.813
g/mL, respectively. The purified fusion pro-
teins was also diluted and then took 100 μL diluted
standard solutions respectively to mix with 2 400 μL
CBB (Coomassie Brilliant Blue), the OD
595
value was
read three times by spectrophotometer using ddH
2
O as
reference.
3.5 Enzymatic activity of Fusion protein
Enzymatic activity of fusion protein was measured by
the method of Vallee and Hoch (Vallee and Hoch,
1955), 150 μL pyrophosphate buffer, 50 μL substrate
solution and 100 μL solution of NAD
+
were mixed to
a cuvette. The cuvette was placed in water bath at
37 for 5
℃
min and then added 10 μL purified fusion
protein solution preheated under the same conditions,
immediately count the time and read the increase of
absorbance at 340 nm every 1min interval in the suc-
cessive 5min until the values of absorbance is stable.
3.6 Resistant analysis of the fusion protein ex-
pressed in
E. coli
Employ Echave’s method to be slightly modified to
analyze the prokaryotic resistance (Echave et al., 2003)
in this reserch. 2.5 mL recombinant strain solution and
its reference respectively cultured overnight until the
OD
600
reached 0.3, then added 22.5 mL LB media
containing 100 μg/mL ampicillin and 25 μg/mL ka-
namycin cultured at 37 . then 0.1
℃
mmol/L IPTG
was added to dilute ten times, The cultured media
were divided into two groups, one group of the
recombinant added H
2
O
2
with a final concentration of
1 mmol/L and the other group added water as control,
the OD
600
values were measured in every 30 min to
calculate the resistant activity.
3.7 Construction of yeast expression vector
The primers were designed based on
LcADH1
se-
quence information, forward primer as: 5'-GGGATCC
ATGTCGACCACAGCT-3' with
Bam
H
Ⅰ
site, and
reverse primer as: 5'-CGTCTAGAACACATCATTG
TTTTTG-3 with
Xba
Ⅰ
site. pMD18T-LjADH1 (with
restriction sites
Bam
H
Ⅰ
and
Xba
) and pYES2
Ⅰ
(Clontech) vectors were digested with
Bam
H and
Ⅰ
Xba
. The products of digestion were recovered to
Ⅰ
ligate into pYES2 vector, the recombinant was named
pYES2-LjADH1. then transformed into the yeast
INVScl
(Clontech) by LiAc/PEG chemical transfor-
mation method (Gietz et al., 1995).
3.8 Resistant analysis of LjADH1 protein expressed
in yeast
The recombinant
INVScI
strains harboring pYES2 and
pYES2-LjADH1 respectively were cultured in liquid
SC-U medium at 30
℃
overnight culture until the
OD
600
reached 0.6. Then diluted them to 10
-1
times,
10
-2
times, 10
-3
times, 10
-4
times
and 10
-5
times, res-
pecttively with YPG medium. Picked up 5 µL strain
solution in the pYES2-LjADH1 and pYES2 medium
in each dilution concentration, dropped on YPG solid
medium which containing 10 mmol/L CuCl
2
, 1.8 mmol/L
NiCl
2
, 100 μmol/L CdCl
2
, 150 μmol/L CdCl
2
and
3.5 mmol/LH
2
O
2
, respectively cultured at 30 for 2 days.
℃
Authors’ contributions
TZ and SKL designed and conducted this experiments. RYL, PTG and DGZ
participated the experiment design and data analysis; XJF is the person who
takes charge of this project, including experiment design, data analysis,
writing and modifying of the manuscript. All authors have read and
approved the final manuscript.
Acknowledgements
This research was supported by Scientific Supporting Project of Ministty of
Science and Technology of China (2007BAD59B05). Authors appreciate Dr
X Zhang from the ASNESC of N FU for her kindly technological supports
and helpful advice on the experiment. Many thanks to two anonymous
reviewers for their useful critical comments and revising advice to this paper.
And also we mentioned some reagent suppliers and sequencing service
providers in this work, that doesn’t mean we would like to recommend or
endorse their products and services.
References
Chervin C., Truett J.K., and Speirs J., 1999, Alcohol dehydrogenase