Hi there,

There are two obvious possibilities :

1) your fusion is shorter than expected (either premature stop codon in the cloned insert (but it's not the case as you have sequenced the full insert) or protein is degraded at C-terminus during expression or extraction)

2) your fusion protein doesn't behave as a 46kD protein on SDS-PAGE for unkown reason (maybe compacted form although SDS treatment or your protein is retaining more negative charges than expected).

Hope this will help.

Nonsense mutation? Is the insert in frame? What strain are you using?

I have checked the sequence and the frame, they are absolutely fine. I am using BL21(DE3) bacterial strain.

It could also be your mw ladder and buffer combination. Some ladders run differently in different types of gel/buffer system. For example check out this page: http://www.taq-dna.com/protein-marker-2c-gel-staining-_112.html. Scroll down and you will see that the same ladder run in Tris-glycine, Bis-tris MOPS and bis-tris MES, yields different molecular weights for each marker. I have had this problem myself once upon a time. I hope this is the problem, because there is none then.

http://www.taq-dna.com/protein-marker-2c-gel-staining-_112.html

Look at this... it might be describing what is happening in your case.

Novagen pET System Manuel, pg 40, Secondary Site Translation Initiation

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0CCEQFjAA&url=http%3A%2F%2Flifeserv.bgu.ac.il%2Fwb%2Fzarivach%2Fmedia%2Fprotocols%2FNovagen%2520pET%2520system%2520manual.pdf&ei=ZoBzVLm4GO-IiwK10IHgDw&usg=AFQjCNFR1oBsOMv6HnJRBwL_AMTjv3SIXQ&sig2=8hLDMFALGdTPamU_lVWvtw

SDS-PAGE is only a relative measure of molecular weight. So other methods, such as MALS and SEC, can also be used if you want a more accurate measurement of your proteins molecular weight.

Did you confirmed molecular weight by MALDI?