Methods and compositions for concentrating secreted recombinant proteins
View Patent ↗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.
1. A preparation of Kluyveromyces cells characterized by a chitinase-negative phenotype wherein the phenotype is the result of a mutation in a chitinase gene expressing secreted chitinase, the preparation when grown for 48 hours being capable of producing substantially the same dry cell mass as wild-type Kluyveromyces cells.
2. A preparation of Kluyveromyces cells according to claim 1 , further capable of expressing and secreting recombinant protein.
3. A preparation of Kluyveromyces cells according to claim 2 , wherein expression is regulated by a LAC4 promoter or modification thereof.
4. A preparation of Kluyveromyces cells according to claim 1 , comprising a shuttle vector, the shuttle vector having a modified Lac4 promoter for expressing a protein in Kluyveromyces while expressing substantially no protein in E. coli.
5. A preparation of Kluyveromyces cells according to claim 2 , wherein the shuttle vector is pKLACI.
6. A preparation of Kluyveromyces cells according to claim 1 , further comprising a culture medium in which the Kluyveromyces is capable of at least one of growth and maintenance, the culture medium further comprising sterilized chitin.
7. A preparation of Kluyveromyces cells according to claim 6 , wherein the sterilized chitin is in the form of magnetized chitin beads.
8. A preparation of Kluyveromyces cells according to claim 1 , wherein the preparation is contained in a vessel, the vessel optionally containing a magnet or in contact with a magnet for reversibly binding the chitin beads.