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Bioscience Methods
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The induced strains were collected by centrifugation
at 4 000 r/min for 20 min and, after discarding the
supernatant, the pellet was frozen at -80 for 2℃ hours,
before completely suspending by first adding 5 mL
suspension liquid and continuing to add suspension
liquid to a final volume of 15 mL. Lysozyme was then
added at 1 mg/mL to digest while shaking for 30 min
in the ice bath.
Conditions for optimun expression were carried out
under the IPTG concentrations of 0.1 mmol/L,
0.5 mmol/L, 1.0 mmol/L and temperatures at 20 ,℃
28 , 37 .℃ ℃ Lysates were collected by centrifugation
at 10 000 r/min at 4 for 40℃ min and 1 mL
supernatant used for preparing samples for
SDS-PAGE detection (Bradford, 1976).
3.5 The purification of His-tag-Cry1Ac22 fusion
protein
Affinity chromatography with Ni2+ -NTA Resin
Column was used to purify the His-tag-Cry1Ac22
fusion protein. Supernatants were collected by super
speed centrifugation and the pellet was transferred
onto the Ni2+ -NTA Resin Column for 2 h. Collect
Recombinant bacterium cells were collected and
cooled in an ice bath for 15 min and then Buffer B
was added to re-suspend the collected pellet. The
suspension was stirred for at least 15 min, (maxmum
60 min) at room temperature until the lytic reaction
was finished. Supernatants were collected by super
speed centrifugation at 10 000 g for 20 to 30 min, then
transferred onto the Ni2+-NTA Resin Column. Buffer
C was initially used to wash the column while
collecting the fractionation liquid for SDS-PAGE
analysis. This was followed by four washings with
Buffer D and a final four washings with Buffer E to
collect the respective liquid fractions for analysis by
SDS-PAGE.
3.6 Bioassary
Second instar larvae of Plutella xylostella were
provided by HITAR (Haide Institute of Tropical
Agricultural Resources). Larvacidal assays followed
the procedures of Xie et al (2010).
Authors’ contributions
ZML and SKL are the persons who executed this research and
prepared the manuscript experiment; YZL partly managed the
project and was involved in data analysis. XJF is the principal
investigator for this project in charge of conducting
experimental design, data analysis and preparing the
manuscript. All authors have read and consent to the final
version of this paper.
Acknowledgements
Our sincere thanks to all the people who have provided their
strong technological support and useful advice during our
experiments including Dr X. Zhang from ASNESC, and Mrs W.
Zhang and L. Xie from HITAR. We greatly appreciate Dr. Phil
Grau, Sr. Entomologist of SynTech Research, for reading and
revising the manuscript. Many thanks to two anonymous
reviewers for their strict criticism on this paper. This work was
supported by the China National Bt Collection Initiative project.
In this paper, mention of trade names or commercial products is
solely for the purpose of providing specific information and
does not imply recommendation or endorsement by authors or
institutes or the University involved in this study.
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