Soluble recombinant protein production
OwnerBrendan McKeown, Christopher Scott +3inventors
InventorsBrendan McKeown, Christopher Scott, Alan McBride, Richard Buick, Jim Johnston
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Abstract
Described is a method of producing a soluble bioactive domain of a protein, the method comprising the step of selecting suitable soluble subunits of a protein and assessing the produced protein for desired activity. The method may comprise the steps of amplifying DNA encoding at least one candidate soluble domain, cloning the amplified DNA into at least one expression vector, using each of said vectors into which the DNA has been cloned to each transfect or transform one or more host cell strains, expressing said DNA in one or more host cell strains, and analyzing expression products from said host cells for solubility.
Claims43
A method of producing a soluble bioactive domain of a protein of interest, the method comprising the step of selecting at least one candidate soluble domain of the protein and assessing the produced protein of each domain for desired activity.
A method of producing a soluble bioactive domain of a protein of interest comprising the steps:
- (a) analysing DNA coding for the protein of interest to identify one or more candidate soluble domains,
- (b) providing oligonucleotide primers to amplify DNA encoding each domain,
- (c) amplifying said DNA using, in parallel, a standard PCR programme using a predicted annealing temperature for the primers;
- (ii) a standard PCR programme using a temperature in the range 48 to 52° C., preferably 50° C., as the temperature for annealin;
- and (iii) a touchdown PCR programme, where the annealing temperature starts at a temperature in the range 62 to 67° C., preferably 65° C., and then gradually decreases to a temperature in the range 48 to 52° C., preferably 50° C., over the subsequent cycles,
- (d) cloning amplified DNA from step (b) into a plurality of different expression vectors,
- (e) optionally screening clones for correct orientation of DNA,
- (f) using each of the vectors of step (d) into which the DNA has been cloned to each transfect or transform a plurality of different host cell strains,
- (g) expressing said DNA in one or more of said host cell strains, and
- (h) analysing expression products from said host cells for solubility.
Use of a sortase gene product as a purification tag.
An expression construct for the production of recombinant polypeptides, which construct comprises an expression cassette consisting of the following elements that are operably linked:
- a) a promoter;
- b) the coding region of a DNA encoding a sortase gene product as a purification tag sequence;
- and c) a cloning site for receiving the coding region for the recombinant polypeptide to be produced;
- and d) transcription termination signals.
A purification tag comprising a sortase gene product.
Dependencies are inferred from the claim text.