HighLevel Expression, Purification, and Characterization of Recombinant Type A Botulinum Neurotoxin - PowerPoint PPT Presentation

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HighLevel Expression, Purification, and Characterization of Recombinant Type A Botulinum Neurotoxin

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(B) Add Elution buffer 30 ml. ???????? 2 ( A) Add wash buffer 15 ml (B) Add Elution buffer 30 ml. Pool A ???????? 1, 2 and Pool B ???????? 1, 2 ... – PowerPoint PPT presentation

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Title: HighLevel Expression, Purification, and Characterization of Recombinant Type A Botulinum Neurotoxin


1
High-Level Expression, Purification, and
Characterization of Recombinant Type A Botulinum
Neurotoxin Light Chain
MS.SAKULRAT PADPROM
2
  • Clostridium botulinum ?

3
  • Method
  • Expression of Recombinant BoNT/A LC
  • Inoculate recombinant BoNT/A LC to 50 ml 2YT
    medium

37 oC, Shake, O/N
Aliquots Freeze -70 oC
Inoculate recombinant BoNT/A LC to 100 ml 2YT
medium
37 oC, Shake, O/N
Add 100 ml culture OD 0.6 to 1 lit 2YT medium
30 oC, Shake ,O/N OD0.6-0.7
4
Add IPTG final 0.5 mM
30 oC, Shake, 15 hrs.
Harvest Cell
Centrifuge 4,000 g, 10 min ,4 oC
Freeze -70 oC
5
  • Purification

Add 50 ml lysis buffer in cell pellet
Sonicate on ice pulse 2 x 1.5 min
(1) Centrifuge 4,000 g, 10 min,4 oC
Add 20 ml lysis buffer
6
Sonicate on ice pulse 2 x 1.5 min
(2)Centrifuge 4,000 g, 10 min,4 oC
Pool (1) (2)
Centrifuge 50,000 g, 1 hr.,4 oC
Bind onto His bed 2 ml
7
???????? 1
( A) Add wash buffer 15 ml
(B) Add Elution buffer 30 ml
???????? 2
( A) Add wash buffer 15 ml
(B) Add Elution buffer 30 ml
8
Pool A ???????? 1, 2 and Pool B ???????? 1, 2
Dialyze O/N with 10 mM PBS pH 8.0
Pool A and Pool B load onto DEAE-A50 column
Equilibrate with same buffer
Fraction for analyze by SDS-PAGE and ELISA Test
9
SDS-PAGE
1.5 ml 0f cell culture and
Centrifuge 4,000 g, 10 min
Resuspended in 300 ul 2x reducing SDS sample
buffer
Heat at 100 oC, 3 min
10
Centrifuge 5,000 g, 15 min
5 ul of the sample were applied to the SDS-PAGE
gel
Detected by Coomassie blue staining
11
Isoelectric Focusing
Endopeptidase Activity
Circular Dichoism Spectroscopy
12
Result
FIG 1. SDS-PAGE analysis of total cell lysate of
E. Coli HB101 containing pBN3 showing the
expression of LC at different induction times
(lane 2-9) ,Supernatant and Pellets of the cell
crude extract were also analyzed (lane 10 and 11)
13
TABLE 1 Purification Scheme of Recombinant BoNT/A
LC
aEstimated from band intensity on
SDS-PAGE. bCalculated based on total protein
content.
14
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16
FIG 4 Cleavage of SNAP-25 by BoNT/A ( ) and
recombinant LC ( )
17
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18
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