1185-pDB.His.MBP-Int

Cloning of pDB.His.MBP-Int(1185)
Purpose: to clone pDB.His.MBP-Int
Date: 2012-5-16 to 2012-5-
Notebook: Xiaojing Wang
Page: 004249
Clone ID: 1185
Experimenters: Xiaojing Wang
Results

digest 406 pET28b-Int with Nde1 + Xho1, --insert
digest V14 pDB.His.MBP with Nde1 + Xho1---> vector

Ligate and transform
Select 6 clones to mini-prep
1185 plasmids are digested by Nde1 + Xho1

1185-1~-6 may be all correct.

Pick 1185-1 to sequence.

Protein Expression

induction, 37 oC 5 hours

Lane	ID	Name	Pi, MW	Induction	Sample	Expression
1	1708	pET28b-scIHF2			S
2	1203	pET42b-scIHF2			S
3	1074	pET28b-IHFab			S
4	1186	pDB.His.MBP-Xis			S
5	406	pET28b-Int			S
6	1185	pDB.His.MBP-Int
7	1708	pET28b-scIHF2	9.60 / 22.8		P	Y
8	1203	pET42b-scIHF2	8.38 / 54.2		P	Y
9	1074	pET28b-IHFab	9.20 / 25.3		P	N
10	1186	pDB.His.MBP-Xis	6.62 / 52.1		P	Y
11	406	pET28b-Int	9.53 / 42.5		P	N
12	1185	pDB.His.MBP-Int	7.33 / 83.7		P	Y
M		PageRuler Plus	250, 130, 00, 70, 55, 35, 25, 15, 10

==

10/21/19

Purification from 2 L culture. Induce at 18 oC o/n, 0.5 mM IPTG

Lane	Sample, fraction #	Note	Note
1	1185, #37	looks like MBP, truncated, maybe int is not expressed	Cell lysed and regrow,
2	1185, #38	looks like MBP, truncated, maybe int is not expressed	truncated, only MBP
3	1078	Ni-->Q, elute using 0.05-->1.5 M NaCl gradient
4	1078	Ni-->Q, elute using 0.05-->1.5 M NaCl gradient
5	1186 , #5	Ni-->Q, elute using 0.05-->1.5 M NaCl gradient	has expression
6	1186, #8	Ni-->Q, elute using 0.05-->1.5 M NaCl gradient	has expression
7	1186, #6	washed from Ni using 30 mM Imidazole	has expression
8	1186, #7	washed from Ni using 30 mM Imidazole	has expression
9	1185, #12	Ni-->Q, elute using 0.05-->1.5 M NaCl gradient
10	1185, #16	Ni-->Q, elute using 0.05-->1.5 M NaCl gradient
11	1185, #17	Ni-->Q, elute using 0.05-->1.5 M NaCl gradient
M	PageRuler Plus	250, 130, 00, 70, 55, 35, 25, 15, 10

Purification of 1185

expression host: Nico21(DE3)plysN, LB with 1X 5052, 0.5 mM IPTG, 17 oC o/n

Lane	ID	Name	Fraction
1	1186	MBP-Xis	Lot 173
2	1186	MBP-Xis	Lot 172
3	1185	MBP (truncated from Int exp)	Lot 171
4	1185	MBP (truncated from Int exp)	Lot 174  elute from Q-SP, fraction 18
5			Lot 174  elute from Q-SP, F 19
6			Lot 174  elute from Q-SP, F 20
7			Lot 174  elute from Q-SP, F 21
8		junk, discard	Lot 174  elute from Q-SP, F25
9		junk, discard	Lot 174  elute from Q-SP, F26
10			Lot 174 FT on Q-SP F7
11			Lot 174 FT on Q-SP F27
M

11/1/2019

Use Nico21(DE3)pRare2pLysS, 15 ml, induced with 1 mM IPTG, 37 oC 5 hours

cre in Nico21(DE3)pLysN, (from freezer, not freshly transformed) 8L, induce 37 oC 6 hours, 0.15% arabinose., use 50 ml Ni column

Lane	1		MW	Expression
1	1078	pET28b-scIHF2, soluble	22.8	N
2	406	pET28b-Int, soluble	42.5	N
3	1185	pDB.His.MBP-Int, soluble	83.7	Y
4	2640	pCold-MBP-(Ent-TEV)-scIHF, soluble	66.9	N
5	2640	pCold-MBP-(Ent-TEV)-scIHF, inclusion body	66.9	Y
6~12	1010	Cre,		could be low
13	1078	pET28b-scIHF2	22.8	N
14	406	pET28b-Int	42.5	N
M		PageRuler Plus