Dear Gaurab Banerjee

I, myself, was in a similar situation few years ago. I have had a small protein expressed from recombinant technology. My intention was to get the protein into solution for structural studies. However, during purification, it has been always localized in insoluble fraction. most of the times, during dialysis it was going ok until the third day. However, at the last minutes when the concentration of urea goes low all of the protein has been precipitated. I do not have enough experience to help you how you do it. However, I would like you to think this way. Even if you fold back your protein into its "native" state, how do you want to prove that this particular state is the native state (and it is not an arbitrary state). The best approach seems to be purify the native protein into your soluble fraction that you do not even need to re-fold your peptide. I did not change my strategy of purification at that time and kept purifying by his-tag. Maybe if you follow some alternative purification options you can get it in a native state and you do not need to fold back it into some state and the consider it as native state. I know that this is not enough answer to your question. It might be a sharing of feeling though.

Sincerely

Mohsen

Dear Gaurab Banerjee

I, myself, was in a similar situation few years ago. I have had a small protein expressed from recombinant technology. My intention was to get the protein into solution for structural studies. However, during purification, it has been always localized in insoluble fraction. most of the times, during dialysis it was going ok until the third day. However, at the last minutes when the concentration of urea goes low all of the protein has been precipitated. I do not have enough experience to help you how you do it. However, I would like you to think this way. Even if you fold back your protein into its "native" state, how do you want to prove that this particular state is the native state (and it is not an arbitrary state). The best approach seems to be purify the native protein into your soluble fraction that you do not even need to re-fold your peptide. I did not change my strategy of purification at that time and kept purifying by his-tag. Maybe if you follow some alternative purification options you can get it in a native state and you do not need to fold back it into some state and the consider it as native state. I know that this is not enough answer to your question. It might be a sharing of feeling though.

Sincerely

Mohsen

You can express eukayotic proteins in bacteria, purify themunder native conditions, crystallize them and study their functions. Since, certain modifications [acetylation, methylation,phosphorylation and glycosylation] are not available in bacteria you may use Pichia expression system. One goal of molecular cloning is to study the molecule individually. You may have to alter certain parameters for expression such as inducible expression, amino-terminal or carboxy terminal tagging to enable a successful purification using stationary matrices. The tagging has to be tested before proceeding to major steps as certain proteins tagged at the amino-terminus may not be biologically active. Similarly, functions of some other proteins may be disturbed by carboxy terminal tagging. There are several biotech companies marketing systems for expression, purification and modification of the recombinant proteins.

For example: The Restriction and Modification enzymes used recombinant DNA research are expressed using their respective cDNAs. Many recombinant proteins are purified and crystallized. Their Crystal structure is studied by X-ray diffraction. All the factors of transcription and translation are expressed and purified. Your countryman who received Nobel prize recently studied the structure of Ribosome using recombinant subunits.

Hope you have enough materials to go through a Google search to retrieve scholarly articles.

Have fun

Gaurav, can you give some more details? Like if you're expressing eukaryotic protein in bacteria?? what cells and vector you are using? and whether you're loosing biological activity right in cell lysate or during purification? maybe some details can help us understand the problem in a better way.

We do a lot of jobs about recombinant protein which fused enzyme molecules. I think the most important is selection of linker. Both long, flexible linker and rigidity linker could help retaining the activity of enzyme in recombinant protein. The characters of the enzyme is the key factor to fuse with the other protein still retaining the activity.

The sucess of modern biotechnology states to express the foreign genes in host cells. However, transcription and translation may not lead to accumulation of fully folded conformationally active protein. It is also well known that proteins accumulate in an insoluble state called inclusion bodies which contains almost pure proteins linked by covalent forces which could be solubilized by strong denaturing agents. The challenge is to convert this into a active and useful product.

Firstly thanks for all the replies. Your answers were helpful in getting some more ideas.

We generally take two approaches. First we try to reduce the concentration of Urea and see for proper folding. Multiple dilutions we use along with the temperature kept low. This is once the 6(His)- Tag is removed.

The second approach is if the His-tag is present we generally try various sequential dilutions of urea to pass while we purify the protein in the column itself. Reducing the concentration serially gets us a better folding.

My question is it possible to get 100% biologically active enzyme this way or am I just wasting my time. My protein of Interest is Methionine aminopeptidase (MetAP1).

Anybody who can send me a related work or with the same protein will be a great help.

Dear Ashutosh,

We are expressing in E.Coli with our own standardized pRSET vector. The expression can also be in Pichia if we change our mind. We went for a gene optimization prior to gene synthesis and then cloned it without error in to our lab standardized vector.

What we are really looking for is a protein folding technique?

Dear Gaurab,

Its possible to get the biological active of enzyme..However, if u r expressing eukaryotic protein in E.coli then its activity is around 80-90% of the source. So, RDT is a boon to modern day molecular biologist. express it in different condition lke optimize the temp....thats low temp, around 24 or even 16 degree if ur using BL21 DE3 or Plys or C41a......even though u r not getting the soluble protein then see u can use some soluble tags like GST or MBP.......though these tags r not useful for HMW proteins.....

First, I would say yes , recombinant enzyme during the heterologous expression can retained the 100% enzyme activity, However it depends upon the mechinary of the host system whether it is capable to transcribe the exact copy of that mRNA sequence, twhich will encode for the actual polypeptide for a certain function without any error, or whether it recognize the genetic codon present in the gene that is cloned, as sometimes codon bias is also a big problem in host system , Addtionally, it is also possible to loose the activity of recombinant enzyme during the processing of the protein after the cell lysis, or during the concentrating a protein after it is secreted extracellulairly into the medium, also sometime protein goes into the inclusion bodies, and threfore may resulted in loss of enzyme activity after refolding it from urea solution, however i will suggest to Gaurav to please, discuss in detail the problem he is facing that may help him in geeting more suggestion. Good luck

yes - Activity can be retail. If activity of your protein after expression is directly correlated with solubility try to use proso server to check whether your protein will be soluble once expressed in ecoli

http://mips.helmholtz-muenchen.de/prosoII/prosoII.seam

Have a look at the amino acid sequence to determine if there are areas that could cause instability. For instance, one can often delete the first 20 or so amino acids without losing any activity and the truncated proteins are often dramatically more stable.

Please remember that part of re-folding may be the need to fully reduce disulfides and then allow them to re-form correctly.

Thanks again for all your feed back.

I have made my mind to proceed the work as a pilot run. All your suggestions are valuable and shall intimate you when I would require more suggestions.

With regards,

Gaurab Banerjee

I think you could seriously consider Aismul's suggestion.Try glycerol or some other polyol perhaps even PEG at low concentratioon, if it doesn't interfere with your enzyme assay. It really depends a lot on the amino acid composition (hydrophobic ?).

Also many enzymes do not suffer dillution well. Has anyone reported assay conditions for your ensyme? Have they noted soubliity problems or protein dillution effects on activity? Unfortunately theory only takes you so far.. You just have to try various approaches.

Best of luck,

-Doug Easton

Yes. There are numerous of publications out there whereby they compare the catalytic efficiency of a recombinant enzyme to that of a wild type. Sometimes, their activities are comparable and sometimes they are not.

It might be good to see what the effects of various treatments describe above have on the activity of the enzyme. Also , if others have been able to isolate recombinant enzyme, it seems logical to try at least a modified version of their protocol.