Modified phosphatases
The invention relates to phosphatases and more in specific to (genetically) modified phosphatases, pharmaceutical compositions comprising (genetically) modified phosphatases and the use of (genetically) modified phosphatases for treating or curing for example sepsis, inflammatory bowel disease or other inflammatory diseases, or renal failure. The invention further relates to a method for producing phosphatases.
1. An alkaline phosphatase, wherein the alkaline phosphatase is isolated or recombinant and comprises:
a human intestinal alkaline phosphatase (ALPI) wherein the crown domain of the human ALPI, corresponding to amino acids 366-430 of SEQ ID NO:2, is replaced with the crown domain of a human placental alkaline phosphatase (ALPP), corresponding to amino acids 366-430 of SEQ ID NO:1,
wherein the alkaline phosphatase has dephosphorylation activity.
2. The alkaline phosphatase of claim 1 , wherein the amino acid sequence of the alkaline phosphatase comprises a glycosylphosphatidylinositol (“GPI”) signal sequence comprising a modification or a deletion in the GPI signal sequence and the modification or deletion results in the alkaline phosphatase being a secreted phosphatase.
3. A method for producing the alkaline phosphatase of claim 1 , the method comprising:
culturing a host cell comprising a polynucleotide encoding the alkaline phosphatase in a medium comprising Zn2+ and
allowing the cell to produce the alkaline phosphatase.
4. The method according to claim 3 , further comprising isolating the alkaline phosphatase.
5. The alkaline phosphatase of claim 1 , produced by a method comprising:
culturing a host cell comprising a polynucleotide encoding the alkaline phosphatase in a medium comprising Zn2+, and
producing the alkaline phosphatase in the host cell.
6. An in vitro/biochemical method for dephosphorylating a substrate in an environment having a Zn2+ concentration lower than 10 uM, the method comprising:
utilizing the alkaline phosphatase of claim 1 to dephosphorylate the substrate.
7. The method according to claim 3 , wherein the host cell is a mammalian cell.
8. The method according to claim 6 , wherein the substrate is an adenosine phosphate.
9. The alkaline phosphatase of claim 1 , wherein the crown domain of the human ALPP is as set forth by amino acids 366-430 of SEQ ID NO:1.
10. The alkaline phosphatase of claim 1 , which comprises the amino acid sequence of SEQ ID NO:5.
11. The alkaline phosphatase of claim 1 , wherein the alkaline phosphatase retains at least 75% of its initial specific activity at a Zn 2+ concentration of about 0.01 μM over a 22 hour period of time.
12. The alkaline phosphatase of claim 1 , wherein the specific activity of the alkaline phosphatase is greater than secreted human placental alkaline phosphatase (sALPP), wherein the specific activity of the alkaline phosphatase and the human sALPP are tested at physiological Zn 2+ levels.
13. The alkaline phosphatase of claim 1 , wherein the specific activity of the alkaline phosphatase is greater than the specific activity of secreted human ALPI, wherein the specific activity of the alkaline phosphatase and the human sALPI are tested at physiological Zn 2+ levels.
14. The alkaline phosphatase of claim 1 , wherein the alkaline phosphatase has improved stability at 56° C. over a 22 hour period compared to human sALPP and sALPI.
15. The alkaline phosphatase of claim 1 , wherein the alkaline phosphatase dephosphorylates an adenosine phosphate or para-nitrophenylphosphate (pNPP).