Regardless of the host expression system, you'll have to isolate your gene and insert it into the appropriate vector. We'll do everything necessary to get your gene amplified, adapted and inserted in-frame with the vector initiation site.
Gene to Protein - Cloning to Protein Purification
From structural biology, drug discovery, lead optimization, high throughput studies (HTS) or characterization of the protein of interest, we can work with you to get going with all your Cloning, Expression optimization, Production to final purification of your protein. We also have extensive experience working with different systems such as E.coli, Baculovirus, Insect and mammalian cell lines and can provide production quantities of your protein in the shortest possible timeframe.
|From gene to protein
|PCR amplification of the gene
||2 weeks||Primer design and gene amplication from the template.Gel picture with the gene of interest run against the molecular weight markers uploaded on the project page. Sample of the PCR product can be sent upon request before the gene is inserted into the desired vector.|
|Subcloning||1-2 weeks||From customer sent plasmid, gene will be amplified by PCR and transfered to the desired expression vector for expression. Insert will be checked by size and sequencing. Sequencing results and the gel picture will be uploaded on the project page.|
||1 week||Check protein expression using Small scale cultures (5ml). Total protein run on SDS-gel against molecular weight markers. Selection of the best expressing colonies for further optimization and scale up.|
|Expression Optimization - Pilot Scale
||1 week||The selected clones will be checked again under 3 different temperature conditions and 2 different induction conditions. Protein yield and characteristics will be determined.There may be an extra charge for this option.|
|2-3 weeks||The identified clones will be used for protein expression. 1 liter cultures will be used for testing different purification methods. Tagged proteins will be first purified using affinity chromatography, followed by tag removal (if desired by client) and the progress checked by SDS-PAGE and western blotting with anti-his tag antibody, where applicable. There may be additional charge for other tags.Polishing will be done by gel-filtration or ion-exchange chromatogrpahy. Native proteins (un-tagged) will be purified by using traditional column chromatogrpahic principles. QC data will be uploaded on to the project page. Process/Method developed for large scale protein purification of desired protein or the developed method is transferred to client.|
|Large Scale Production||2-3 weeks||We can scale up protein production from 5 liters to 400 liters. Cultures will be spun down and pellet/cell paste in desired buffer can be either sent to client or used in protein purification.|
|Purification of Protein||2-3 weeks||The expressed protein will be purified to near homogeniety using affinity column or by utilizing traitional column chromatographic principles. QC data will be uploaded on the project page and the purified protein in the final buffer will be sent to the customer.|