large scale protein purification - (Sep/03/2015 )

Hi all, can somebody suggest bulk scale protein purification protocols for proteins produced extracellulary in pichia pastoris? Are there special protocols for food industry enzyme purification?

thanks

Crude isolation usually starts with salting out using ammonium sulfate in increasing concentrations. This can be followed in many ways, such as ionic columns such as monoQ, or size exclusion columns. If you can add a tag, then 6His tagged proteins can be isolated on nickel binding columns, and eluted with imidazole. I'd recommend you read the free protein purification handbooks published by GE.

and here's the link to the webpage to download the ge handbooks:

handbooks

Thanks... and what could be the maximum amount of proteins recovered by sephadex columns?

that depends on the size of the sephadex column and the volume of the sample.

information on how to determine this with your setup is in the handbook.

keep in mind that sephadex is for gel filtration. nothing should bind under proper conditions.

The protein is extracellular so your first problem is concentrating the protein; using salt precipitation could give problems with waste disposal at industrial scales; ion exchange is a good method but the limited availability (and high cost) of food grade IE-resins might restrict your options.

If you are working commercially, how pure do you need the protein to be? Assuming your yeast and all the starting components are GRAS, the optimal purification cost/price on an industrial scale is often achieved with simple centrifugation followed by filtration.

Ultrafiltration/diafiltration is a good way to begin a purification of a heterologous protein secreted from Pichia. It allows you to change the buffer, pH and reduce the big initial fermentor volumen in order to work afterwards in lab-scale columns.