Method for protein isolation in anoxic conditions
AssigneeMSD Oss B.V.
InventorsPeter Van Der Meijden, Gijsbert Willem Karel Van Dedem, Michel Hendrikus Maria Eppink, Roeland Wilhelmus Wassenaar
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Abstract
The present invention relates to a method for the isolation of proteins that comprise disulfide bonds in their native conformation. Essentially, a method of the present invention makes use of reducing agents such as β-mercaptoethanol or dithiothreitol in protein isolation methods obsolete. A method of the present invention is particularly suitable for the isolation of precursor proteins such as proinsulin from recombinant cells.
Claims5
A method for isolating a recombinant protein that comprises disulfide-bonds in its native conformation, said method comprising the steps of:
- a. maintaining a culture of recombinant prokaryotic host cells containing an expression cassette which directs the expression of the recombinant protein under culture conditions suitable for protein expression;
- b. harvesting and disrupting the prokaryotic host cells thereby producing a crude cell extract and;
- c. isolating the recombinant protein from the cell extract of step b);
- wherein steps b)-c) are performed under essentially anoxic conditions and the recombinant protein recovered in step c) is isolated in its native conformation and biologically active form without the use of reducing agents;
- and wherein the recombinant protein is present as inclusion bodies in the prokaryotic host cells.
Dependencies are inferred from the claim text.