Hi Ankita: I think that you should repeat the cloning using another vector. One possibility is bind your gene and tag by PCR and use the same vector. In order to purify a protein by affinity you need a tag or antibody against you gene.

In the absence of tags, you can use anion or cation exchange resin (depending on the PI of your protein) as a first step, followed by gel filtration. Another option is to engineer a hexa his on the amino terminal using PCR thsi will give you an added affinity step and should not interefer with protein function.

You can use pFASTBAC-Ht or Baculodirect C or N-term Ht baculoviral vector system to clone your gene of interest, which has C or N terminal polyhistidine tag for purification using Ni-affinity resin.

As the posts above, I'd say it depends what are you using your proteins for. If you don't need your protein to be a complete replica as in vivo, then I would do as the posts suggest, and add a tag by changing the fragment from plasmid or by adding the tag by PCR. If that's not the case, then you should design a purification protocol. For this is pretty useful to know the denaturation temperature, the solubility in the presence of diverse chaotropic agents, the isoelectric point, etc, as you can use this info to eliminate non-wanted contaminants. For chromatographic purification, as Sarju said, an ion exchange media is pretty good as an initial approach, followed by a more specific chromatographic step (has your protein any affinity in particular?, have you any purified antibodies against it?). Hope this helps a bit.

Also, if you are worried about the purification tag affecting downstream applications, there are cleavable purification tags that can be removed by enzymatic reactions. Basically you bind your protein using the purification tag to the respective resin, then wash away non-specifics, then cleave the purified protein from the resin, leaving the purification tag behind.

Thank you all for your valuable suggestions. I would go for ion exchange chromatography for its initial purification and I need this protein for some enzymatic assays. This protein does not have antibodies against it.

Often our clients don't want a tag even if it is cleavable because there's always residual non cleaved. This is to satisfy requirements for human therapeutics.

You may want to try ammonium sulphate precipitation cuts before you go to your ion column. This involves precipitating proteins with increasing percentages of ammonium sulphate. The advantage is that one can remove a lot of non target proteins in the lower percentage cuts.

Clear the lysate by centrifugation.

Ammonium sulphate precipitation. Identify the cut with your protein.

Gel filtration of this to narrow down to proteins of similar size

anion and/or cation exchange

I would use standard purification techniques. Besides ion exchange, there is hydrophobic chromatography, phenyl sepharose, affinity chromatograph, and dye columns to name a few.

1. Hydrophobic chromatography such as phenyl, octyl, hexyl sepharose is used by putting the protein in high salt and then eluting with a high to low salt gradient.

2. Affinity chromatography depends on what your protein likes to bind to. So if it is an enzyme, you cn attach inhibitorsor substrates to a column. For popular enzymes and proteins, things like GTP, ATP, NADPH, are commercially available. One can by the "pre activated" resin and attach most anything. The protein is competed off by soluble ligand/inhibitor/substrate, etc.

Also if your protein is glycosylated, there is affinity resins using concannavslin A and wheat germ agglutinin.

3. For dye columns, kits can be purchased and testing is done empirically.

4. I forgot to mention there is also hydroxyapatite and Heparin columns. These work by charge and other factors.

All the above answers seem to got the idea, so I will not tell any new techniques; instead I'll recommend you to check this android phone app

https://play.google.com/store/apps/details?id=com.agbooth.pp_phone&hl=es

You can "train" here to have an idea how some of the techniques that the colleagues here are telling you to use (I would recommend this to they too, for teaching or just for fun).