Methods and compositions for concentrating secreted recombinant protein
AssigneeNew England Biolabs, Inc.
InventorsChristopher H. Taron, Paul A. Colussi
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Abstract
Methods and compositions are described that relate to obtaining concentrated preparations of secreted recombinant proteins. These proteins are expressed in the form of fusion proteins with a chitin-binding domain (CBD). The fusion proteins are capable of being concentrated in the presence of chitin. Also described is: a shuttle vector that includes a modified LAC4 promoter; a chitinase-negative host cell; a CBD capable of eluting from chitin under non-denaturing conditions; and sterilized chitin, which can be optionally magnetized for facilitating recovery of recombinant protein.
Claims19
A method of obtaining a concentrated preparation of a secreted recombinant protein, comprising:
- (a) transforming host expression cells with a shuttle vector containing a DNA encoding a fusion protein comprising a chitin-binding domain (CBD) and a target protein;
- (b) expressing the fusion protein in the host expression cells and secreting the fusion protein therefrom;
- and
- (c) binding the secreted fusion protein to a preparation of chitin by means of the CBD, the fusion protein being capable of elution into a desired buffer volume under non-denaturing conditions so as to obtain a concentrated preparation of the secreted recombinant protein.
A method of obtaining a concentrated preparation of a secreted recombinant protein, comprising:
- (a) providing a shuttle vector, wherein the shuttle vector (i) is capable of integration into the genome of a yeast host cell, and (ii) contains a DNA, the DNA encoding a fusion protein comprising a chitin binding domain and a target protein;
- (b) transforming a chitinase deficient host expression cell with the shuttle vector for expressing the fusion protein in the yeast host expression cell and secreting the fusion protein therefrom;
- and
- (c) binding the secreted fusion protein to a preparation of chitin by means of the CBD so as to obtain the concentrated preparation of secreted protein.
Dependencies are inferred from the claim text.