Methods and compositions for concentrating secreted recombinant protein
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 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.
2. A method according to claim 1 , wherein the DNA encoding the target protein in the vector is transcriptionally silent in E.coli and transcriptionally active in the host expression cells.
3. A method according to claim 1 , wherein the shuttle vector has a modified LAC4 promoter.
4. A method according to claim 1 herein the shuttle vector is pKLAC1.
5. A method according to claim 1 , wherein the host expression cells are chitinase-deficient cells.
6. A method according to claim 2 , wherein the host expression cells are yeast cells.
7. A method according to claim 4 , wherein the yeast cells are a single species selected from a Kluyveromyces, a Yarrowia, a Pichia, a Hansenula, and a Saccharomyces species.
8. A method according to claim 6 , wherein the host expression cells are Kluyveromyces.
9. A method according to claim 8 , wherein the Kluyveromyces is a Kluyveromyces marxianus variety fragilis or lactis.
10. A method according to claim 1 , wherein the host expression cells are in a culture, such that the fusion protein is secreted into the culture medium.
11. A method according to claim 10 , further comprising: adding the chitin to the culture medium during cultivation.
12. A method according to claim 11 , wherein the chitin is sterile.
13. A method for according to claim 1 or 10 , wherein the chitin is added to the mixture after cultivation.
14. A method according to claim 1 or 12 , wherein the chitin is in a form selected from a coating, a colloid, a bead, a column, a matrix, a sheet and a membrane.
15. A method according to claim 1 or 12 , wherein the chitin is a chitin bead and the bead is either porous or non-porous.
16. A method according to claim 15 , wherein the chitin bead is magnetized.
17. A method according to claim 16 , wherein recovering the fusion protein bound to the chitin in step (d) further comprises applying a magnetic force.
18. A method according to claim 1 , wherein the binding of the fusion protein to chitin is reversible such that the fusion protein can be released from the chitin under non-denaturing conditions that differ from the conditions for binding.
19. 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.