CNBr cleavage of MBP-fusion protein?

pip at VENT.NEB.COM pip at VENT.NEB.COM
Wed May 12 09:15:11 EST 1993


>To anyone using the pMal protein fusion and purification system from NEB or 
>anyone familiar with cyanogen bromide cleavages:
>
>Are any of you using cyanogen bromide to cleave the fusion protein?
>
>I have been told by somebody that the maltose (10mM)in the column elution 
>buffer can cause incomplete cleavage in the cleavage step.  It was recommended
>that I dialyze before the cleavage step.
>
>Can anyone confirm this observation?
>
>I would appreciate any feedback I can get on this.  Thanks!
>
>Angie. 

Angie

We haven't used CnBr cleavage yet, because none of our fusions that give
problems with factor Xa have had unique methionines. I've attached the
sequence of MBP* from pMAL-c2 so you can predict the sizes of the 7 CnBR
peptides it should produce. 
I think the business with factor Xa being inhibited by 10 mM maltose may
have come from a poorly-worded sentence in the first pMAL manual. 10 mM
maltose doesn't inhibit factor Xa digestion. 
I'm assuming you have a problem with factorXa, since you're considering
CnBr. Is your problem lack of cleavage, or the presence of secondary sites
in your protein? Low concentrations of SDS (0.005-0.1%) can sometimes help
with the former (e.g., Ellinger et al., 1991, Virol 180,811-3).
 

Mbp2.Pep  Length: 387  February 13, 1992  

       1  MKIEEGKLVI WINGDKGYNG LAEVGKKFEK DTGIKVTVEH PDKLEEKFPQ

      51  VAATGDGPDI IFWAHDRFGG YAQSGLLAEI TPDKAFQDKL YPFTWDAVRY

     101  NGKLIAYPIA VEALSLIYNK DLLPNPPKTW EEIPALDKEL KAKGKSALMF

     151  NLQEPYFTWP LIAADGGYAF KYENGKYDIK DVGVDNAGAK AGLTFLVDLI

     201  KNKHMNADTD YSIAEAAFNK GETAMTINGP WAWSNIDTSK VNYGVTVLPT

     251  FKGQPSKPFV GVLSAGINAA SPNKELAKEF LENYLLTDEG LEAVNKDKPL

     301  GAVALKSYEE ELAKDPRIAA TMENAQKGEI MPNIPQMSAF WYAVRTAVIN

     351  AASGRQTVDE ALKDAQTNSS SNNNNNNNNN NLGIEGRLD


 PEPTIDESORT of: MBP2.pep  check: 9659  from: 1  to: 389

Molecular weight =   42709.89     Residues =    389
Average Residue Weight = 109.794     Charged =  -11
Isoelectric point =  4.86
Extinction coefficient =  64720

Residue           Number      Mole Percent     ..

A = Ala            44           11.311
B = Asx             0            0.000
C = Cys             0            0.000
D = Asp            25            6.427
E = Glu            28            7.198
F = Phe            15            3.856
G = Gly            31            7.969
H = His             3            0.771
I = Ile            23            5.913
K = Lys            36            9.254
L = Leu            32            8.226
M = Met             7            1.799
N = Asn            32            8.226
P = Pro            21            5.398
Q = Gln             9            2.314
R = Arg             6            1.542
S = Ser            15            3.856
T = Thr            19            4.884
V = Val            20            5.141
W = Trp             8            2.057
Y = Tyr            15            3.856
Z = Glx             0            0.000

A + G              75           19.280
S + T              34            8.740
D + E              53           13.625
D + E + N +  Q     94           24.165
H + K + R          45           11.568
D + E + H + K + R  98           25.193
I + L + M + V      82           21.080
F + W + Y          38            9.769


_______________________________________________________________
Paul Riggs       	       	       	508-927-5054 x254
New England Biolabs     	        fax: 508-921-1350
32 Tozer Rd     	       	       	
Beverly  MA  01915-5599 	       	Internet: riggs at vent.neb.com





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