It would be helpful if you could rephrase the question and provide more details. It appears as if there are two problems here, 1) "something wrong happens during expression" and 2) "finally, the protein molecular weight is lower than our target". Is that so?

In any case, look in the literature to see if someone else has expressed this enzyme. If it has been done before, did the authors have the same problem? That can help you narrow down possible causes.

Also, can you please clarify how did you determine MW? SDS-PAGE? Mass spectrometry? Gel filtration?

If MW is indeed wrong, the most usual culprits (I am assuming expression in E. coli due to your "competent cells" comment) are proteolytic degradation and the presence of cryptic translational inititation sites that are more efficient than the one provided by the vector. None of those have easy, straightforward solutions.

Hope it helps!

Expression of Cellulose 1,4-beta-cellobiosidase:

This enzyme catalyses the hydrolysis of 1,4‐β glycoside linkages in cellulose and releases cellobiose G2 from the non-reducing ends.

This enzyme was extensively studied, purified, biochemical characterized, cloned and expressed from eukaryotic cells and usually from fungi; and usually this enzyme protein are induced and secreted and also glycosylated.

Question: What kind of organism have you used to prepare the DNA gene (eukaryotic or bacterial) and what type of cell have you used to clone and express your CBH gene?

I believe that the manner of your work is to clone and express CBH fungal gene in bacteria as E coli and you know that in this case no glycosylation was occurred so the MW (Molecular Weight) the purified recombinant protein is lower than the native glycosylated protein.

Yet I want to ask you if your cloned CBH is still active?

Comment: May be you have to clone the CBH gene in eukaryotic system as Yeast: Pichia or Saccharomyces system and so you could prepare and active glycosylated recombinant CBH protein.

In that program I could send you a protocol to express your CBH gene in P. pastoris :

Heterologous expression and biochemical characterization of a novel thermostable Sclerotinia sclerotiorum GH45 endoglucanase in Pichia pastoris.

Haifa Chahed, Mohamed Boumaiza, Aymen Ezzine and M. Nejib Marzouki (2017)

International Journal of Biological Macromolecules DOI: 10.1016/j.ijbiomac.2017.08.062

Best regards

i also suggest rephrase your question and more details , only you have MW issues or other. agree to Alejandro Martin .

Hello  Dears,

First thank you so much for giving the detailed solution for my problem. yes you are right I have to include the details of my experiment to facilitate your answers.

MW was totally wrong, I had determined it by SDS-PAGE and Mass spectrometer, at the time my cloned CBH is not active .

these are my work steps:

• Source: Caldicellulosiruptor bescii DSM 6725

•I Isolated this gene and ligated to pEASY blunt E1 vector.

•Transformation of recombinant plasmid vector to E.coli Trans1-T1 phage resistant chemically competent cells

•After transforming to Trans1 T1 competent cells, I got some clones

•Colony PCR.

•Sequence verification of recombinant plasmid vector

•Expression using E.coli BL21(DE3) , C43.

lately, I am trying to clone the CBH gene in Eukaryota system using Pichia pastoris GS115 .

I ligated this to pPICZα A then, transformed it to Trans1 T1 competent cells, I got some clones, did PCR but no success, I am still trying .

Dear Alejandro,

Thank you so much, could you please explain this point more ( proteolytic degradation and the presence of cryptic translational inititation sites ), do you have any suggestion to avoid this?

Re: cryptic translational initiation.

The problem is that the trasnlational initiation signals of eukaryotes (as is your case; yours is a fungal enzyme, right?) and prokaryotes differ. The translation of eukaryotic mRNA usually starts at the first AUG found in 5' to 3' direction, assuming the mRNA is properly capped, etc., whereas in prokaryotes, translational initiation can take place at any AUG that is preceded by a sequence stretch able to base pair with the right parts of the 16S rRNA subunit (that's the famous Shine-Dalgarno sequence, or the Ribosome Binding Site or RBS in more modern parlance).

Now, the problem is that the requirements for a functional RBS are not that stringent, so if a eukaryotic gene has several internal AUG codons, there is a small, but non-zero probability that one of them will be preceded by a sequence able to act as a functional RBS in bacteria. In that case, that AUG codon will also be used as a start codon, giving rise to a smaller protein in addition to the full-length species. Mind you, this is rare, I explained this first just to get it out of the way.

The second problem is obvious: you are expressing a eukaryotic enzyme in E. coli, so 1) It will not be glycosylated, 2) if the native structure depends on the presence of disulfide-bonded cysteins, it will never fold correctly in vivo. Both can leave a protein susceptible to degradation by proteases from the host (which, to boot, are different from those of the environment -i.e. organism- in which the protein is expressed natively). Proteolytic degradation is suspect number 1 in cases like this, unfortunately (of course, I am assuming that it is taking place during culture, is this the case? Degradation during purification is a totally different problem...)

Solutions? In the case of proteolytic degradation in vivo, you can tinker with culture conditions (lower temperatures sometimes work), and if you can identify the cleavage site and you are able to tinker with the sequence of the protein (usually you cannot) you could try site-directed mutagenesis to change the sequence of the target site withouth altering the biological activity of the protein. Sometimes the only sensible solution is to express in a different cellular compartment or to switch to a different heterologous host.

Dear Alejandro,

Really I appreciated your simple and detailed solutions but I would like to inform you that, my gene origin is Prokaryotic (Caldicellulosiruptor bescii DSM 6725)

Did your protein form inclusion body? If so then you have to do more work to make it become active form.

Dear Muhammad,

In fact, I don't know if it forms or not. I will see.

@ Hamed,

Still, the same solutions apply. Try lower temperatures, try switching to a K12 strain (some of the SHuffle strains from NEB are K12 rather than B strains, for instance), try periplasmic rather than cytoplasmic expression. If you did MS then you know where the protein is being cleaved. You can change the P1 or P1' residue by site-directed mutagenesis if they are not involved in catalysis or part of the active site.

Hope it helps!

Dear Prof.Alejandro,

Thank you so much for giving me such valuable answers.