MBP fusion protein... - (Sep/01/2006 )
Hi,
I am doing expresion of a MBP-fusion protein in DH5a, however, a protein band at about 42kd was found, which means, my fusion protein has been cut and only the MBP left.... What can I do?........
no reply......................
Your protein might not be cleaved from the MBP fusion, sometimes if your target protein is difficult to express, only MBP might then be expressed.
I am doing expresion of a MBP-fusion protein in DH5a, however, a protein band at about 42kd was found, which means, my fusion protein has been cut and only the MBP left.... What can I do?........
hello,
try expressing your protein in an expression strain like BL21 or BL21 Rosetta. while DH5 alpha can be used for protein expression, it's primarily a cloning strain. also, you may have had vector contamination, and aren't expressing your protein as a fusion- just MBP alone. if you haven't done so already, make sure your plasmid contains your insert and that it's in-frame by sequencing, or by using PCR w/ the appropriate primers. alternatively, you can do a restriction digest, but this will tell you nothing about the reading frame.
good luck
I am doing expresion of a MBP-fusion protein in DH5a, however, a protein band at about 42kd was found, which means, my fusion protein has been cut and only the MBP left.... What can I do?........
Thanks,
The target protein is actually a fragment peptide of a large protein. Reading frame is correct, sequece correct... but anyway I will try BL cells.
may be your (MBP+fusion protein) is too big so it stuck in the well,,how big is your protein?
The protein fragment is about 22kd, (about 200aa). MBP is 42kd..