Methods for identifying proteins with starch phosphorylating enzymatic activity
View Patent ↗The present invention relates to a method for identifying proteins involved in the phosphorylation of starch and nucleic acids which code for such proteins. The present invention further relates to plant cells and plants which exhibit an altered activity of a protein which can be identified using the method according to the invention. Plant cells and plants of this type synthesise a modified starch. The present invention therefore also relates to the starch synthesised by the plant cells and plants according to the invention as well as to methods for the manufacture of this starch and to the manufacture of starch derivatives of this modified starch.
1. A method for identifying a protein which has an elevated binding activity towards phosphorylated alpha-1,4-glucans, compared to non-phosphorylated alpha-1,4 glucans, comprising
a) incubating protein extracts in preparations separate from one another with
i. phosphorylated alpha-1,4-glucans and
ii. non-phosphorylated alpha-1,4-glucans,
b) dissolving proteins specifically bound to the
i. phosphorylated alpha-1,4-glucans from step a) i and
ii. proteins specifically bound to the non-phosphorylated alpha-1,4-glucans from step a) ii
in preparations separate from one another and
c) identifying proteins which exhibit an elevated binding activity towards phosphorylated alpha-1,4-glucans used in step b) i, compared to non-phosphorylated alpha-1,4-glucans used in step b) ii.
2. An isolated protein obtained by the method of claim 1 , wherein the isolated protein is an OK1 protein and wherein said OK1 protein comprises SEQ ID No.: 5.
3. A method for identifying a nucleic acid molecule coding for a protein which exhibits alpha-1,4-glucan phosphorylating enzymatic activity,
a) identifying a protein by a method according to claim 1 ,
b) determining amino acid sequences coding for the protein identified according to step a) and
c) identifying nucleic acid molecules using the amino acids determined according to step b).
4. The method according to claim 3 , wherein nucleic acid oligonucleotides based on the amino acid sequence determined according to step b) are manufactured to identify said nucleic acid molecule according to step c).
5. The method for identifying a nucleic acid molecule coding for a protein which exhibits alpha-1,4-glucan phosphorylating enzymatic activity, comprising
a) identifying a protein by a method according to claim 1 ,
b) producing antibodies which react specifically with the protein identified according to step a) and
c) identifying nucleic acid molecules using the antibodies produced according to step b).
6. A method for identifying a protein which exhibits alpha-1,4-glucan phosphorylating enzymatic activity and requires phosphorylated alpha-1,4-glucans as substrate, comprising
a) incubating protein extracts with phosphorylated alpha-1,4-glucans,
b) dissolving proteins specifically bound to the phosphorylated alpha- 1 , 4 -glucans from step a),
c) incubating proteins obtained according to step b) respectively with
i. ATP and phosphorylated alpha-1,4-glucans and
ii. ATP and non-phosphorylated alpha-1,4-glucans in preparations separated from one another,
d) examining the respective alpha-1,4-glucan obtained after incubation in step c) i or step c) ii for introduction of further phosphate groups and
e) identifying proteins which in the incubation preparation according to c) i have introduced significant quantities of phosphate groups into alpha-1,4-glucans and in the incubation preparation according to c) ii have introduced no significant quantities of phosphate groups into alpha-1,4-glucans.
7. The method according to claim 6 , wherein the protein with alpha-1,4-glucan phosphorylating enzymatic activity uses phosphorylated starch as substrate.
8. The method according to claim 7 , wherein the protein with alpha-1,4-glucan phosphorylating enzymatic activity originates from a plant.
9. An isolated protein obtained by the method claim 6 , wherein the isolated protein is an OK1 protein, and wherein said OK1 protein requires phosphorylated α-1,4 glucans as a substrate and comprises SEQ ID No.: 5.
10. A method for identifying a nucleic acid molecule coding for a protein which exhibits alpha-1,4-glucan phosphorylating enzymatic activity, comprising
a) identifying a protein by a method according to claim 6 ,
b) determining amino acid sequences coding for the protein identified according to step a) and
c) identify nucleic acid molecules using the amino acids determined according to step b).
11. The method according to claim 10 , wherein nucleic acid oligonucleotides based on the amino acid sequence determined according to step b) are manufactured to identify said nucleic acid molecule according to step c).
12. The method for identifying a nucleic acid molecule coding for a protein which exhibits alpha-1,4-glucan phosphorylating enzymatic activity, comprising
a) identify a protein by a method according to claim 6 ,
b) producing antibodies which react specifically with the protein identified according to step a) and
c) identify nucleic acid molecules using the antibodies produced according to step b).
13. The isolated protein of claim 2 , wherein the protein comprises SEQ ID No.: 4.
14. The isolated protein of claim 9 , wherein the protein introduces phosphate monoester bonds into the C-3 position of a glucose molecule of a P-α-1,4 glucan.