4 replies to this topic

[deepbluedna]

After expressing my protein with the IPTG induction in BL21 cells. I got not activity of my protein, but there is a band at the correct MW. Then I sent my protein for N-term sequencing in case, after I got the sequence back I found out that the sequence match part of my protein, which means 15 amino acids are missed after methionine. I wonder if it could be caused by the expression of the purification process? Did this ever happen to anyone here? I started a second time expression and got the right band again but no activity.  Problem for me:(
Need help here. Thanks in advance

[perneseblue]

how is the plasmid? Is the DNA sequence correct?

[swanny]

deepbluedna, on Feb 4 2009, 09:20 AM, said:

After expressing my protein with the IPTG induction in BL21 cells. I got not activity of my protein, but there is a band at the correct MW. Then I sent my protein for N-term sequencing in case, after I got the sequence back I found out that the sequence match part of my protein, which means 15 amino acids are missed after methionine. I wonder if it could be caused by the expression of the purification process? Did this ever happen to anyone here? I started a second time expression and got the right band again but no activity.  Problem for me:(
Need help here. Thanks in advance

My first guess is that you have a Met about 15 residues in from your start, right? If so, the transcription/translation machinery has jumped past the true start codon.  If not, there is something very strange going on.

[deepbluedna]

swanny, on Feb 3 2009, 07:18 PM, said:

deepbluedna, on Feb 4 2009, 09:20 AM, said:

After expressing my protein with the IPTG induction in BL21 cells. I got not activity of my protein, but there is a band at the correct MW. Then I sent my protein for N-term sequencing in case, after I got the sequence back I found out that the sequence match part of my protein, which means 15 amino acids are missed after methionine. I wonder if it could be caused by the expression of the purification process? Did this ever happen to anyone here? I started a second time expression and got the right band again but no activity.  Problem for me:(
Need help here. Thanks in advance

My first guess is that you have a Met about 15 residues in from your start, right? If so, the transcription/translation machinery has jumped past the true start codon.  If not, there is something very strange going on.

No, there is no Met ahead of that sequence. Basically the expression start with the Met and jump 15 AAs to keep expressing.

[deepbluedna]

perneseblue, on Feb 3 2009, 06:13 PM, said:

how is the plasmid? Is the DNA sequence correct?

The DNA sequencing is correct.