Sequence homology with some Mycobacteria

• hypothetical protein MkmsDRAFT_2415 [Mycobacterium sp. KMS]

• hypothetical protein MjlsDRAFT_2401 [Mycobacterium sp. JLS]

• hypothetical protein MvanDRAFT_0830 [Mycobacterium vanbaalenii PYR-1]

Lab meeting. December 9, 2006Jae ho, LEE

1. PCR of the homology sequence of hypothetical protein in Mycobacterium smegmatis

2. Expression of the His tagged Rv3676 of Mycobacterium smegmatis

Small culture on 4ml 7H9 + 0.2% glucose + HygromycinB media Large culture on 200 ml 7H9 + 30% CO + hygromycinB media

Small culture on 4ml 7H9 + 0.2% glucose + HygromycinB media Large culture on 200 ml 7H9 + 0.2% glucose + hygromycinB media

3. Expression of the His tagged CutR of Mycobacterium smegmatis

Small culture on 4ml 7H9 + 0.2% glucose + HygromycinB media

4. Detection of protein interacting with Orf4 using His-tag in Mycobacterium smegmatis

pMsm orf4

Transformation of E. coli strain BL21

Small culture large culture on 400ml LB-ampicillin media

Induction condition

10 ml LB-ampicillin media

0.2 mM IPTG (Isopropyl-β-D-thiogalactoside)

37 for 2 hrs , 18 for 12 hrs ~ 17 hrs℃ ℃

0.5 mM IPTG

37 for 3 hrs , 18 for 12 hrs ~ 16 hrs℃ ℃

Mycobacterium smegmatis wild type

small culture on 4 ml SMB + 0.2% glucose media

large culture on 400 ml SMB + 30% CO media

pMsmOrf45595 bp

bla

lacI His-tagged Orf4

orf4

pMsmOrf4-T/NcoI and XbaI

Nco I (341)

Xba I (1246)

Induction of his-tagged Orf4 with IPTG

10% denaturing polyacrylamide gel 0.2 mM IPTG , 37 for 2 hrs , 18 for 12 hrs ~ 17 hrs℃ ℃

Lane 1: protein markerLane 2: crude extract before treatment of IPTGLane 3: crude extract after treatment of IPTG; 12 hrsLane 4: crude extract after treatment of IPTG; 13 hrsLane 5: crude extract after treatment of IPTG; 14 hrsLane 6: crude extract after treatment of IPTG; 15 hrsLane 7: crude extract after treatment of IPTG; 16 hrsLane 8: crude extract after treatment of IPTG; 17 hrs

(kDa)

187

127

80

52

42

27

1 2 3 4 5 6 7 8

33.5 kDa : His-tagged Orf4

Induction of his-tagged Orf4 with IPTG

10% denaturing polyacrylamide gel 0.5 mM IPTG , 37 for 3 hrs , 18 for 12 hrs ~ 16 hrs℃ ℃

Lane 1: protein markerLane 2: crude extract before treatment of IPTGLane 3: crude extract after treatment of IPTG; 12 hrsLane 4: crude extract after treatment of IPTG; 13 hrsLane 5: crude extract after treatment of IPTG; 14 hrsLane 6: crude extract after treatment of IPTG; 15 hrsLane 7: crude extract after treatment of IPTG; 16 hrs

(kDa)

187

127

80

52

42

27

1 2 3 4 5 6 7

33.5 kDa : His-tagged Orf4

Induction of his-tagged Orf4 with IPTG

(kDa)

187

127

80

52

42

27

1 2 3

10% denaturing polyacrylamide gel Lane 1: protein markerLane 2: crude extract before treatment of IPTGLane 3: crude extract of after treatment of IPTG

0.2 mM Isopropyl-β-D-thiogalactoside 18 , 14 hrs ℃

33.5 kDa

Induction of his-tagged Orf4 with IPTG

1 2 3 4 5 6 7 8 9 10 (kDa)

187

127

80

52

42

27

10 % denaturing polyacrylamide gel Lane 1: protein markerLane 2: crude extract Lane 3: purified crude extractLane 4: wash1Lane 5: elution 3Lane 6: elution 5Lane 7: elution 7Lane 8: elution 9Lane 9: elution 11Lane 10: elution 13

0.2 mM Isopropyl-β-D-thiogalactoside 18 , 15 hrs ℃

35kDa

Purification of the CO dehydrogenase of Mycobacterium sp. strain JC1

1 2 3 1 2 3 1 2 3 1 2 3

Figure 1. Western blotting of the CO-DH of Mycobacterium sp. strain JC1

1. Mycobacterium sp. strain JC1 wild type

2. Mycobacterium sp. strain JC1 cutA mutant with pNBV1 JC1 CutA

3. Mycobacterium sp. strain JC1 cutA mutant competent cell

CO Glucose Glucose CO

pNBV1/EcoR¥´ + pMsmOrf4/Sph¥°8379 bp

HygR

pAL5000

pMsmOrf4/sph¥°

Nco I (445)

Nco I (4037)

pNBV1/EcoR¥´ + pMsmOrf4/SphI reverse8379 bp

HygR

pAL5000

reverse pMSMorf4

Nco I (445)

Nco I (3409)

4787 bp

3592 bp

5415 bp

2964 bp

Co-Immuno-Precipitation of the CutR of M. smegmatis

(kDa)

187

127

80

52

42

27

Lane 1: protein marker Lane 2: IP crude extract Lane 3: IP product

FIGURE 3 . Co-Immuno-Precipitation of His tagged MSM CutR protein

in 10% SDS gel.

80 kDa

55 kDa

19 Kda

15 Kda

1 2 3

60 kDa

50 kDa

34 kDa

30 kDa