Page 8 - BM 2010 Vol.1 No.2

Bioscience Methods
BM 2010, Vol.1, No.2 http://bm.sophiapublisher.com
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expressed in E.coli exists in the form of inclusion
bodies. These are a kind of toxic protein existing in
the host cell which reduces the concentration of the
foreign protein and alleviates poisoning the host cells,
increases the amount of protein expression. We think
that purifying Cry1Ac22 protein using urea as
denaturant will little affect the structure and function,
which will be helpful to further study the physic-
chemical characteristics and in vitro toxicity.
3 Materials and Methods
3.1 Strains, plasmids and chemicalls
Details of the strains and plasmids used in this
experiment are listed in Table 1. Taq DNA polyrm-
erase, dNTP, DNA ladder marker and λDNA /HindⅢ
marker were bought from DEMei Biotechnology Co.
Ltd; SalⅠ and Bam HⅠ were from TaKaRa Firm.
Ni2+ -NTAn resin is the production of Novagen
Company.
3.2 Cry1Ac22 PCR amplification
A pair of primers was designed for amplifying
Cry1Ac22 gene based on the sequence deposited in
GenBank with accession no Eu282379, while
BamH andⅠ Sal restriction enzymic cutting sites
Ⅰ
were introduced into the termination of the primers.
The forward primer is as GGA TCC ATG GAT AAC
AAT CCG AAC ATC, and the reverse primer as GTC
GAC TGA GTT TGC ATG AGA CTA TTC. The
underlined letters refer to the restriction enzymic
cutting sites of Bam HⅠ and SalⅠ. The target gene
was PCR amplified using plasmid DNA from Bt
W015-1 under the conditions of PCR amplification as
follows: Pre-denaturation at 95℃ for 5 min, followed
by 30 cycles (94 ℃ 30 s, 54℃ 30 s and 72℃ 30 s),
and finished at 72℃ for 5 min.
3.3 Construction procedures of recombinant expre-
ssion vector
The enzymic cutting vector pQE30 and amplified
Cry1Ac22 were mixed at a ratio of 3 moles to 1 mole
in the ligation reaction mixture and deionized water
was added to a final volume 20 μL. Prior to
transformation, the mixture was held overnight in an
ice bath at 16 to make pQE30℃ -Cry1Ac22. 4 μL
ligation product were placed in an ice bath with
defrosting M15 competent cells for 30 min, prior to
heat shock at 42 for 90℃ s, and then 400 μL LB
medium were added and the culture was shaken for 1
hour at 37 .℃ 100 μL of the transformed liquid was
placed on LB screening plates with 100 μg/mL
ampicillin and 50 μg/mL kanamycin at 37℃ for
overnight culture. Single colonies were picked and
used for PCR to identification of positive recombinant
clones.
3.4 Expression of fusion protein His-tag-Cry1Ac22
induced and optimized
A positive single bacterial colony was incubated in 5 mL
LB liquid culture medium which was shaken
overnight at 37 ,℃ and then diluted with fresh medium
at a ratio of 1:50 to increase the culture. Recombinant
protein was then induced by adding 1.0 mmol/L IPTG
at 37 when℃ the OD600 reached about 0.8. 1 mL
samples of culture medium were collected every 30 min.
Table 1 Strains and plasmids used in this research
Strains and plasmids Characterization Origin References
pMD18-T AmpR This lab
pQE30 AmpR, T5 expression vector This lab
E. coli M15 AmpR , Nals , Strs, Rif s, Thi-,Lac-, Ara+ , Gal+, Mtl-, F-, RecA +,
Uvr+, Lon + with KanaR This lab
E. coli JM109 E. coli recA1, supE44, endA1, hsdR17, gyr A96, relA1
thi (lac△ -proAB) This lab Sambrook et al., 2002
Bt W015-1 Wild strain of B. thuringiensis This lab Xie et al., 2010