Page 6 - BM 2010 Vol.1 No.2

Bioscience Methods
BM 2010, Vol.1, No.2 http://bm.sophiapublisher.com
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Figure 2 Identification of the positive colony by PCR
Note: 1~6: Tested transformants individually picked; M:
λDNA/HindⅢ marker
Figure 3 The expression of 6×His-Cry1Ac22 analyzed by
SDS-PAGE
Note: M: PR1500 Protein Marker; 1~6: Lysate collected at the
time of 0 min, 30 min, 60 min, 90 min, 120 min, 180 min after
IPTG induction; Arrow indicates the expressed 6×His-
Cry1Ac22 fusion proteins
28℃ based on the results of SDS-PAGE (Figure 4).
1.2 Expression of fussion protein in E.coli M15
We conducted an experiment for the expression of
Cry1Ac22 inclusion protein under conditions of
120 r/min shaking culture at 28℃ and induced by IPTG
1.0 mmol/L for 3 h, 6 h, 9 h, 12 h to observe the
difference in protein expression among the trans-
formants (Figure 5). The results showed that the amount
of the expressed proteins was gradually increased with
the length of ITPG induction. The different
transformants exhibited their different expression
levels under the fixed culture condition induced for
12 h (Figure 6).
1.3 Purification of the fusion protein
We purified the inclusion proteins expressed in E.coli
using the urea-denaturation procedures. Bacterial
lysate (before purification) and fractionated peak
eluent (after purification) which both contained target
proteins were collected and analyzed by SDS PAGE.
The target protein band presented in the range of
about 133 kD indicating that the fusion protein would
be the dominant component in the profile of whole
proteins in E.coli. The purity of the protein
fractionated with Ni2+-NTA resin is about 80%
(Figure 7). The concentration of the protein was
detected to be 1 177 μg/mL.
1.4 Larvacide assay
Engineered bacterial lysate cultured for 20 hours and
purified fusion protein were diluted and sprayed on
Chinese cabbage leaves. Second instar larvae of
diamondback moth, Plutella xylostella were introduced
onto the leaves and held in an incubator at 25 . The℃
results showed that all larvae on treated leaves were
dead after 30 hours, whereas the control larvae,
normally, meaning that the engineered bacteria and its
purified protein are toxic to the lepidopteran (data not
shown).
2 Discussion
Making a prokaryotic fusion gene construct is a
common and effective way to express an exogenous
gene in E.coli. The prokaryotic expression vector
pQE30 we used in this research is the most popular
plasmid vector, having the characteristics of small
molecular size (3.4 kb) and easy manipulation. It also
has excellent features in its plasmid structures such as
the replicon and ampicillin encoding gene from
pBR322, the intense T5 promoter and the purifying
tag that encodes six histidines (The QIA expressionist.
2001).
E.coli M15 is the host strain that specificly matche
with the pQE30 vector to express foreign protein.
Using this strain can express exogenous gene to
generate 6×His tagging inclusion protein. Fusion
protein expressed in E.coli M15 usually has good
stability and is not easily degraded by bacterial
protease and the expressed inclusion proteins are
easily fractionated by Ni2+-NTA resin affinity chroma-
tography (Huang et al., 2008).