IMMOBILIZED PROTEINS AND METHODS AND USES THEREOF
The invention relates to the field of covalently attaching proteins to a substrate, particularly to methods of immobilizing proteins by posttranslationally modifying a cysteine residue of said protein through the addition of functional groups. The invention also relates to biological molecules used in such techniques, including proteins, and detection methods and kits that utilize such immobilized proteins, such as a microdevice or “protein chip”, a high-throughput screening device, and for the microscopy of proteins on a surface.
1 . A method of covalently immobilizing a protein, comprising:
a. posttranslationally modifying a cysteine residue of said protein through the addition of functional groups, and
b. immobilizing said protein by ligation of said functional groups to a substrate;
wherein said protein comprises one or more of a soluble protein or a solubilized protein.
2 . The method of claim 1 , wherein said cysteine is in a C-terminal CaaX motif of a soluble protein.
3 . The method of claim 2 , where X is A, S, M, L or Q.
4 . The method of claim 1 , wherein said posttranslational modification of a cysteine includes alkylation of the sulfhydryl moiety comprising one or more of catalysis of a farnesyl analog with protein farnesyltransferase and catalysis of a geranylgeranyl analog with protein geranylgeranyltransferase.
5 . The method of claim 4 , where said functional group comprises one or more of an azide, an alkyne, an alkene, a ketone and an alkoxyamine.
6 . The method of claim 4 , wherein said farnesyl analog is selected from the analogs depicted in FIG. 9 .
7 . The method of claim 1 , wherein said functional group comprises one or more of an azide and an alkyne.
8 . The method of claim 1 , wherein said substrate comprises one or more of a silicon surface selected from the group consisting of silicon, glass, SiO 2 , and silicon nitride, a polymer surface comprising one or more of polymer beads and polymer films, a gel surface comprising one or more of an agarose gel and an acrylamide gel, and a metal surface comprising one or more of platinum, gold, silver, copper, zinc sulfide, cadmium selenide, zinc sulfide-capped cadmium selenide, titanium dioxide, aluminum, aluminum oxide, opal films, and ceramics.
9 . The method of claim 8 , wherein said substrate comprises one or more of an azide-derivatized glass surface, an alkyne-derivatized glass surface, and a phosphine-derivatized glass surface.
10 . A method of covalently immobilizing a protein, comprising:
a. alkylating the sulfhydryl moiety in a C-terminal CaaX motif of a protein through a catalysis selected from the group consisting of catalysis of a farnesyl analog with protein farnesyltransferase and catalysis of a geranylgeranyl analog with protein geranylgeranyltransferase, resulting in the addition of a functional group, and
b. immobilizing the derivatized protein of (a) to a substrate comprising one or more of an azide-derivatized surface, an alkyne-derivatized surface and a phosphine-derivatized surface;
wherein said protein comprises one or more of a soluble protein and a solubilized protein.
11 . The method of claim 10 , where X is A, S, M, L or Q.
12 . The method of claim 10 , where said functional group comprises one or more of an azide, an alkene, an alkyne, a ketone, or an alkoxyamine.
13 . The method of claim 10 , wherein said farnesyl analog is selected from the analogs depicted in FIG. 9 .
14 . An isolated protein comprising a soluble protein having a non-native C-terminal CaaX motif in which the cysteine has been posttranslationally modified, resulting in the addition of a functional group and wherein said protein has been immobilized to a substrate by ligating the functional group to said substrate.
15 . The protein of claim 14 , where X permits alkylation of the cysteine residue by analogues of farnesyl diphosphate or geranylgeranyl diphosphate.
16 . The protein of claim 14 , wherein said posttranslational modification of a cysteine includes alkylation of the sulfhydryl moiety comprising one or more of catalysis of a farnesyl analog with protein farnesyltransferase and catalysis of a geranylgeranyl analog with protein geranylgeranyltransferase
17 . The protein of claim 14 , where said functional group comprises one or more of an azide and an alkyne.
18 . The protein of claim 16 , wherein said farnesyl analog comprises the structure
19 . The protein of claim 14 , wherein said substrate comprises one or more of a silicon surface selected from the group consisting of silicon, glass, SiO 2 , and silicon nitride, a polymer surface comprising one or more of polymer beads and polymer films, a gel surface comprising one or more of an agarose gel and an acrylamide gel, and a metal surface comprising one or more of platinum, gold, silver, copper, zinc sulfide, cadmium selenide, zinc sulfide-capped cadmium selenide, titanium dioxide, aluminum, aluminum oxide, opal films, and ceramics.
20 . The protein of claim 14 , wherein said substrate comprises one or more of an azide-derivatized glass surface, an alkyne-derivatized glass surface, and a phosphine-derivatized glass surface.