Purification of a functionally active protein kinase from bacteria

Andrew J Ippolito aji97 at acsu.buffalo.edu
Mon Apr 24 21:54:35 EST 2000


Dear readers,
    I am a very frustrated 3rd year graduate student that has been
attempting unsuccessfully to purify a functionally active GST-fusion protein
kinase expressed via pGEX from DH5-alpha e.coli.  The protein has been
published as a functionally active autophosphorylating kinase which also
phosphorylates several protein substrates.
    I am using identicle stocks of DNA to transform competent bacteria that
a former postdoc has used to successfully purify the protein.  She has since
left our lab, and we have been unable to reproduce her protocols.  I have
tackled the problem from all different sides, all to no avail.  Here is the
current protocol she insists works; if anyone has any comments on it, I will
appreciate it greatly :)

1.  After transformation select a colony (or several) and prepare overnight
culture (5 ml)
2.  Grow 150 ml culture to OD595 to .6
3.  Induce with 1mM IPTG for 2 hours.
4.  Spin bacteria down for 10 minutes at 5k refridgerated.
5.  Resuspend pellet in 1 ml of Buffer:  50mM HEPES 7.4, 5 mM EDTA, .1%
NP-40, with .5 mM PMSF and 5 ul of aprotinin added fresh.
6.  Freeze/thaw 3x by dry ice/37C water bath.
7.  Spin 10'x13k refridgerated.
8.  To pellet add 500 ul 6M Guanidine:HCl in TBSt as above.  Resuspend.
9.  15' ice.
10.  Spin 10'x13k refridgerated.  Dilute sup with 5 ml above buffer + 300 ul
glut. beads in         1:1 slurry with above buffer.
11.  30' cold room, rocking.
12.  Wash beads in 5-10 ml above buffer with inhibitors 10'x3 times.
13.  Wash beads 2x in kinase buffer (25mM HEPES 7.4, MgCl2 5mM, MnCl 10mM,
NaCl     10mM, 5% glycerol)
14.  Assay via incubation of 10 ul beads with 10 uCi [32P]g-ATP in kinase
buffer at 37C         for 30 minutes.

according to the postdoc, this works.  Not for me.  And it is growing VERY
frustrating!  Can anyone help?  I thank the list in advance for any input.

Sincerely,

Andrew Ippolito
RPCI

we have tried denaturing and nondenaturing conditions (she denatures with 6
M guanidine:HCl in Tris-buffered saline w/ .5% tween20)






More information about the Proteins mailing list