Cell-free expression of protein kinase a for rapid activity assays

Abstract

Functional protein analysis often calls for lengthy, laborious in vivo protein expression and purification, and can be complicated by the lack of stability of the purified protein. In this study, we demonstrate the feasibility of a simplified procedure for functional protein analysis on magnetic particles using cell-free protein synthesis of the catalytic subunit of human cAMP-dependent protein kinase as a HaloTag((R)) fusion protein. The cell-free protein synthesis systems provide quick access to the protein of interest, while the HaloTag technology provides efficient, covalent protein immobilization of the fusion protein, eliminating the need for further protein purification and minimizing storage-related stability issues. The immobilized cPKA fusion protein is assayed directly on magnetic beads and can be used in inhibitor analyses. The combination of rapid protein synthesis and capture technologies can greatly facilitate the process of protein expression and activity screening, and therefore, can become a valuable tool for functional proteomics studies.

Keywords: HaloTag; PKA; cell-free expression; in vitro translation; kinase; magnetic particles; protein immobilization.

Figure 1

Schematic of the Protein Analysis Process. Cell-free expression of the cPKA fusion protein was confirmed by western blot analysis and binding to a fluorescent HaloTag ligand. Kinase activity was measured after immobilization from cell-free reactions in the presence and absence of cPKA inhibitors. The “bead” in the figure represents the HaloLink magnetic resin and the “HT” refers to the HaloTag protein at the amino-terminus of the cPKA protein.

Figure 2

Features of Cell-Free Expression Vectors. The cPKA gene was cloned into Flexi Vectors specific for cell-free expression. All of the vectors possessed the HaloTag gene to generate synthesis of a fusion protein with the tag at the N or C-terminus. Elements depicted include the promoters for T7 and SP6 polymerases (T7 and SP6), untranslated regions (UTRs) located at the 5’ and 3’ of the protein coding regions in pF25-derived vectors pF25 N HT and pF25 C HT, a synthetic poly(A) site and the T7 polymerase terminator sequence (T7t). pFN19 and pFC20 plasmids directed expression in RR and WG. pF25 N HT and pF25 C HT templates were used with the ICE system, while S30 reactions used the pFN18 plasmid. The sequences are not drawn to scale.

Figure 3

Protein Detection. A) Western Blot with anti-PKA antibody. One μl of each protein synthesis reaction was loaded per lane. The arrow points to the expressed cPKA fusion protein, which has a predicted molecular weight 74 kDa. Lane 1, MW markers; Lane 2, recombinant bovine heart PKA catalytic subunit; Lane 3, RR no DNA; Lane 4, RR pFN19; Lane 5, RR pFC20; Lane 6, ICE no DNA; Lane 7, ICE N-terminal fusion; Lane 8, ICE C-terminal fusion; Lane 9, WG no DNA; Lane 10, WG pFN19; Lane 11, WG pFC20; Lane 12, S30 no DNA; Lane 13, S30 pFN18. Proteins of unknown identity were reproducibly detected by immunoblot in the S30 reaction (see Lane 12). B) Fluorimage of the fusion protein fluorescently labeled with HaloTag TMR ligand. Each lane contains the equivalent of 1 μl of protein expression reaction. The arrow points to the expressed cPKA fusion protein, which has a predicted molecular weight 74 kDa. Lanes 1 and 2, purified GST-HaloTag protein (MW 62 kDa); Lane 3, RR no DNA; Lane 4, RR pFN19; Lane 5, RR pFC20; Lane 6, ICE no DNA; Lane 7, ICE N-terminal fusion; Lane 8, ICE C-terminal fusion; Lane 9, WG no DNA; Lane 10, WG pFN19; Lane 11, WG pFC20; Lane 12, S30 no DNA; Lane 13, S30 pFN18. Free, unbound HaloTag TMR ligand is visible at the bottom of the gel.

Figure 4

Detection of cPKA Fusion Proteins Expressed in RR Before and After Immobilization. A) Western Blot Analysis. The amount of fusion protein before and after immobilization was determined by gel electrophoresis followed by immunoblotting with antibody specific for PKA at a 1:1000 fold dilution. A volume equivalent to 1 μl of the protein synthesis reaction was loaded per lane. Lanes 1 and 2: no DNA template negative control reaction; Lanes 3 and 4: pFN19-cPKA; Lanes 5 and 6: pFC20-cPKA. The starting materials from the TNT reactions are in lanes 1, 3 and 5, and the post-immobilization supernatants (“flowthroughs”) are in lanes 2, 4 and 6. B) HaloTag TMR Ligand Binding Assay. The starting material and post-immobilization supernatants were incubated with HaloTag TMR Ligand as indicated in the Experimental Section. A volume equivalent to 1 μl of the starting reaction was loaded per lane in the same lane order listed in Panel A. A comparison of protein levels in lanes 3 and 4, and lanes 5 and 6, is made to determine binding of the fusion protein to the magnetic beads. The arrow points to the expressed cPKA fusion protein.

Figure 5

Inhibition of HaloTag-cPKA expressed in Wheat Germ Extract. cPKA fusion protein expressed from pFN19 in Wheat Germ extract was immobilized onto magnetic beads and assayed for activity using the fluorescent ProFluor PKA Assay, in the presence of the kinase inhibitors H-89, PKI and staurosporine. The final concentration of inhibitor in the reaction was 10 μM. Two amounts of particle solution were used, 10 μl and 20 μl. The assay was also done with rPKA at two concentrations. As negative controls the kinase was incubated with Kinase Buffer (Buffer), DMSO (1% final concentration) or an inhibitor U0126 for an unrelated kinase. Each inhibitor was tested in duplicate and the average fluorescence is depicted. The bars represent the signal from each of the duplicate wells (i.e. the range).

Figure 6

Inhibition of HaloTag-cPKA expressed in Insect Cell Extract. HaloTag-cPKA protein was expressed from the pF25 N HT plasmid in the TNT T7 Insect Cell Extract. After immobilization, 5 μl of particle suspension were added per well of the 96 well plate. Reactions containing positive control human rPKA (0.001 units, 2.3 ng) were also performed. The final concentration of all compounds was 10 μM. Control reactions contain only kinase buffer (KB) or DMSO (1% final concentration). All reactions were done in triplicate. The average fluorescent signal is plotted and the error bars represent ± 1 S.D.

Figure 7

Dependence of the Kinase Signal on ATP. Control reactions for the ProFluor PKA Assay were done in the absence of ATP to confirm the dependency of the signal on ATP and the lack of signal interference by components in the reactions. This figure shows the results for 1% DMSO and 10 μM H-89 in 1% DMSO with both recombinant rPKA and cell-free expressed cPKA fusion protein expressed in insect cell extract from the pF25 N HT vector. The average of triplicate wells is plotted with error bars representing ± 1 S.D.