Methods of preparing cysteine variants of human recombinant IL-11
Disclosed are cysteine variants of interleukin-11 (IL-11) and methods of making and using such proteins in therapeutic applications.
1. A method for preparing an isolated, biologically active recombinant human interleukin-11 (IL-11) cysteine mutein, the method comprising the steps of:
(a) obtaining an Escherichia coli ( E. coli ) host cell transformed with a DNA encoding a an IL-11 cysteine mutein fusion protein, wherein said fusion protein comprises a chitin binding domain, an intein domain and the IL-11 cysteine mutein, wherein the N-terminus of the IL-11 cysteine mutein protein is covalently joined to the C-terminus of the intein domain;
(b) growing the E. coli host cell under conditions that cause the host cell to express the IL-11 cysteine mutein fusion protein in a soluble form in the E. coli cytoplasm;
(c) lysing the cells;
(d) separating insoluble proteins from the soluble proteins comprising the IL-11 cysteine mutein fusion protein;
(e) isolating the IL-11 cysteine mutein fusion protein from the soluble fraction of the cell lysate obtained from step (d) by chitin affinity column chromatography; wherein the IL-11 cysteine mutein fusion protein binds the chitin affinity column;
(f) exposing the IL-11 cysteine mutein fusion protein bound to the chitin affinity column of step (e) to a buffer containing a reducing agent under conditions wherein said reducing agent causes cleavage of the IL-11 cysteine mutein protein from the intein domain; and
(g) washing the column with a buffer to collect the cleaved IL-11 cysteine mutein protein;
and wherein the IL-11 cysteine mutein comprises:
(i) at least one cysteine residue substituted for an amino acid in IL-11 (SEQ ID NO:17) selected from the group consisting of: P22, G23, P24, G27, and A162;
(ii) at least one cysteine residue substituted for an amino acid in IL-11 (SEQ ID NO:17) selected from the group consisting of: P24, P25, E38, L39, D69, L72, S74, T77, A114, S117, E123, A148, and S165, and further comprises deletion of amino acid P22 in IL-11 (SEQ ID NO:17);
(iii) at least one non-native cysteine residue which has been added following the C-terminal amino acid of IL-11 (SEQ ID NO:17);
(iv) at least one non-native cysteine residue which has been added following the C-terminal amino acid of IL-11 (SEQ ID NO:17), and further comprises deletion of amino acid P22 in IL-11 (SEQ ID NO:17);
(v) deletion of one or more amino acids in IL-11 (SEQ ID NO:17) selected from the group consisting of: deletion of amino acid P22, deletion of amino acids 22-26 and deletion of amino acids 22-29; or
(vi) at least two cysteine substitutions, wherein a cysteine residue is substituted for an amino acid in IL-11 (SEQ ID NO:17) selected from the group consisting of P25 and T77, P25 and S117, P25 and S165, P25 and P24, D69 and T77 and A162 and S165 and further comprises deletion of amino acid P22 in IL-11 (SEQ ID NO:17).
2. The method of claim 1 , further comprising purifying the cleaved IL-11 cysteine mutein protein by column chromatography.
3. The method of claim 2 , wherein the column chromatography is ion-exchange column chromatography.
4. The method of claim 3 , wherein the ion-exchange column chromatography is S-Sepharose column chromatography.
5. The method of claim 3 , wherein the ion-exchange column chromatography is Q-Sepharose column chromatography.
6. The method of claim 4 , wherein the IL-11 cysteine mutein protein is passed through a Q-Sepharose column using conditions such that the IL-11 cysteine mutein protein does not bind the Q-sepharose column prior to isolating the IL-11 cysteine mutein protein using S-Sepharose column chromatography.
7. The method of claim 1 , wherein the reducing agent in step (f) is selected from the group consisting of dithiothreitol, beta-mercaptoethanol, TCEP, cysteamine, reduced glutathione and cysteine.
8. The method of claim 1 , wherein the reducing agent in step (f) is dithiothreitol.
9. The method of claim 1 , wherein step (b) occurs at room temperature.
10. The method of claim 1 , wherein step (f) of exposing comprises exposing the IL-11 cysteine mutein fusion protein to the reducing agent at room temperature.
11. The method of claim 1 , wherein the IL-11 cysteine mutein comprises at least one cysteine residue substituted for an amino acid in IL-11 (SEQ ID NO:17) selected from the group consisting of: G23, P24, G27 and A162 and further comprises deletion of amino acid P22 in IL-11 (SEQ ID NO:17).
12. The method of claim 1 , wherein the IL-11 cysteine mutein comprises at least one cysteine residue substituted for an amino acid selected from the group consisting of: P24, P25, E38, L39, D69, L72, S74, T77, A114, S117, E123, A148, and S165, and further comprises deletion of amino acid P22 in IL-11 (SEQ ID NO:17).
13. The method of claim 1 , wherein the IL-11 cysteine mutein is modified with a cysteine-reactive moiety.
14. The method of claim 13 , wherein the cysteine-reactive moiety is a cysteine-reactive polyethylene glycol (PEG) selected from the group consisting of a maleimide-PEG and a vinylsulfone-PEG.
15. The method of claim 13 , wherein the modified IL-11 cysteine mutein is isolated from unmodified IL-11 cysteine mutein.
16. The method of claim 15 , wherein the modified IL-11 cysteine mutein is isolated from unmodified IL-11 cysteine mutein by column chromatography.
17. The method of claim 15 , wherein the modified IL-11 cysteine mutein is isolated from unmodified IL-11 cysteine mutein by S-sepharose column chromatography.