TY - JOUR
AU - Sueyoshi, Noriyuki
AB - Myelin basic protein (MBP) is one of the major components of central nervous system myelin and has multiple sites for protein phosphorylation. Therefore, it has been widely used as a substrate for in vitro assays of various protein kinases. In this study, to obtain more efficient substrates for protein kinase assays than commercially available MBP from bovine brain, we produced various recombinant MBPs using Escherichia coli expression systems. Three splice isoforms of mouse MBP were expressed in E. coli and successfully purified using a new protocol consisting of HCl extraction, urea treatment and affinity purification with HiTrap Chelating HP column. The recombinant MBP isoforms thus obtained served as more efficient substrates for protein kinases than MBP isolated from bovine brain. To generate an even better substrate for protein kinase assays, we produced a hybrid protein composed of two different MBP isoforms connected in tandem, designated TandeMBP. TandeMBP was readily expressed in E. coli and could be purified by the newly developed simple procedure. TandeMBP was phosphorylated by various Ser/Thr protein kinases more efficiently than the other MBP isoforms. Taken together, TandeMBP will become a powerful tool for in vitro assays to analyse various protein kinase activities. In vitro kinase assay, kinase substrate, myelin basic protein, phosphorylation, protein kinase Protein kinases regulate a wide variety of cellular processes such as cell proliferation, differentiation, apoptosis, stress responses and neuronal functions (1). Therefore, an array of protein kinases with different substrate specificities have been reported (2). Among these kinases, multifunctional protein kinases such as MAP kinases, cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinases (CaMK) show broad substrate specificities and can phosphorylate both physiological and non-physiological substrates. The functional roles of protein kinases in various signalling pathways have been studied in vivo and in vitro using several approaches. For characterization of protein kinases in vitro, protein substrates such as myelin basic protein (MBP), casein and histones have frequently been used for assays, because they are known to be preferentially phosphorylated by many protein kinases. Especially, MBP has been widely used for in vitro kinase assays and its phosphorylation sites by Ser/Thr kinases and Tyr kinases have been identified (3–7). MBP is one of the major constituents of the myelin sheath in the central nervous system. The 18.5-kDa MBP is the most predominant isoform in the mammalian brain and shows some characteristic features. First, bovine MBP contains 31 basic amino acid (Lys+Arg) residues and is a highly basic protein with a pI of ∼10.6. Second, MBP is a hydrophilic protein with a relatively low content of hydrophobic amino acids. Third, MBP contains a large number of Ser/Thr residues. Fourth, MBP is susceptible to various post-translational modifications such as deimination, methylation and phosphorylation (8). Based on its characteristic properties, MBP can be classified as an intrinsically unstructured protein and alters its conformation depending on the environment (9). In aqueous solution, MBP appears to behave as a randomly coiled protein with only a small amount of the ordered structure (10). The extended structure of MBP may make it an excellent substrate for various protein kinases (11). Many researchers have obtained commercial MBP for use as a substrate in in vitro kinase assays. The commercially available MBP is a purified preparation from bovine brain and is mainly composed of 18.5-kDa isoform, which is known to be the major component of central nervous system myelin (8). Although MBP has often been used as a substrate for protein kinases, the availability of other MBP isoforms or mutant MBPs as protein kinase substrates remains unclear. In the present study, we attempted to obtain more efficient substrate for in vitro kinase assays than the commonly used natural MBP. First, we obtained three isoforms of mouse MBP: isoform 5, isoform 6 and isoform 8. Judging from their amino acid sequences, MBP(iso5), MBP(iso6) and MBP(iso8) corresponded to 18.5-, 17- and 14-kDa MBP, respectively (12, 13). We prepared recombinant mouse MBP isoforms devoid of any post-translational modifications using Escherichia coli expression systems. The purified MBP isoforms, MBP(iso5), MBP(iso6) and MBP(iso8), served as more efficient substrates for several Ser/Thr protein kinases than the commercial MBP from bovine brain. Furthermore, we produced a unique hybrid MBP, designated TandeMBP, which was a fusion protein of MBP(iso5) and MBP(iso6) connected in tandem. As TandeMBP served as the most efficient substrate for various Ser/Thr protein kinases, it will become a useful tool for analysis of kinase activities in in vitro studies on a variety of protein kinases. Materials and Methods Materials Bovine serum albumin and bovine MBP were purchased from Sigma-Aldrich. [γ-32P]ATP (111 TBq/mmol) was purchased from PerkinElmer. A HiTrap Chelating HP column was obtained from GE Healthcare Bio-Sciences. Escherichia coli BL21(DE3), RosettaI(DE3) and RosettaII(DE3) cells and plasmid pET23a(+) were from Novagen. A horseradish peroxidase-labelled anti-mouse IgG+A+M antibody was obtained from Cappel. The 5′-RACE first-strand cDNA was synthesized from mRNA isolated from mouse brain with Superscript II reverse transcriptase using a SMARTer™ RACE cDNA Amplification Kit (Clontech). Catalytic subunit of PKA was purified from bovine heart as described previously (14). Construction of plasmids To generate expression plasmids for mouse MBP isoforms, the following primers were used for PCR with a mouse brain cDNA library as a template: sense primer (5′-AAA GCT AGC ATG GCA TCA CAG AAG AGA CCC TCA-3′) and antisense primer (5′-AAA GCG TCT CGC CAT GGG AGA TC-3′). The NheI (underlined)-XhoI (double-underlined) fragment was inserted into the NheI-XhoI sites of pET-23a(+) to generate pET-mMBP(iso5), pET-mMBP(iso6) and pET-mMBP(iso8). For TandeMBP, the cDNAs of mouse MBP(iso5) and MBP(iso6) were amplified by PCR with primer sets of 5′-AAA GCT AGC ATG GCA TCA CAG AAG AGA CCC TCA-3′ and 5′-AAA GCG TCT CGC CAT GGG AGA TC-3′ (underline and double underline show NheI and BamHI sites, respectively) or 5′-AAA ATG GCA TCA CAG AAG AGA CCC TCA-3′ and 5′-AAA CTC GAG GCG TCT CGC CAT GGG AGA TC-3′ (underline and double underline show XhoI and BamHI sites, respectively). The PCR fragments were digested with NheI and BamHI or BamHI and XhoI, and ligated into the NheI/XhoI sites of pET-23a(+) to generate pET-TandeMBP. Expression and purification of recombinant MBPs For expression of mouse MBP(iso5), pET-mMBP(iso5) was introduced into E. coli RosettaI(DE3) and cultured in medium A (LB medium containing 100 µg/ml ampicillin and 34 µg/ml chloramphenicol) at 37°C until the A600 reached 0.6, and then isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM. After a 12-h incubation at 18°C, the cells were harvested by centrifugation. For expression of MBP(iso6) and TandeMBP, pET-mMBP(iso6) and pET-TandeMBP were each introduced into E. coli BL21(DE3) and cultured as described above. For expression of mouse MBP(iso8), E. coli RosettaII(DE3) cells carrying pET-mMBP(iso8) were cultured in medium A at 37°C to an A600 of 0.6, and then cultured at 37°C for 6 h in the presence of 0.1 mM IPTG. Escherichia coli cells expressing the recombinant MBP isoforms were harvested from 200 ml of culture by centrifugation, and the cell pellets were suspended in 10 ml of 0.2 M HCl. The cells were disrupted by sonication and the resultant homogenates were shaken at 4°C for 16 h. After extraction of the MBP isoforms with the acidic solution, insoluble materials including cell debris were removed by centrifugation (20,000g for 10 min). The supernatants were neutralized with 1 M NaOH and dissolved in 8 M urea solution (final volume of 20 ml). Each sample containing a His6-tagged protein was loaded onto a HiTrap Chelating HP column (1 ml) pre-equilibrated with buffer A (20 mM Tris-HCl pH 7.5, containing 150 mM NaCl and 0.5% (v/v) Tween 40). The column was then sequentially washed with 10 ml of buffer A, 10 ml of buffer A containing 20 mM imidazole and 10 ml of buffer A containing 50 mM imidazole. Subsequently, the column was eluted with buffer A containing 200 mM imidazole. The purified fractions were pooled, dialysed against buffer A containing 2 mM 2-mercaptoethanol, and stored at −30°C until use. Expression and purification of protein kinases To generate an expression plasmid for mouse Dyrk1A, the following primers were used for PCR with a mouse brain cDNA library as a template: sense primer (5′-G TCT AGA ATG CAT ACA GGA GGA GAG ACT TCA G-3′) and antisense primer (5′-G CGA GCT AGC TAG CTA CAG GAC TCT GTT GC-3′). The XbaI (underlined)-XhoI (double-underlined) fragment was inserted into the NheI-XhoI sites of pET-23a(+). The expression plasmid was introduced into E. coli BL21(DE3) and cultured in LB medium containing 100 µg/ml ampicillin at 37°C until the A600 reached 0.6, and then IPTG was added to a final concentration of 0.1 mM. After a 20-h incubation at 25°C, the cells were harvested by centrifugation. The recombinant Dyrk1A was purified from the soluble fraction using the HiTrap Chelating HP column as described above. Recombinant casein kinase 1 (CK1) (15), constitutively active doublecortin-like protein kinase (DCLK) (16), MAP kinase/extracellular signal-regulated kinase 2 (ERK2) (17), PKL01 (18), CaMKIδ (19) and CaMKK (19) were expressed and purified as described in the cited reports. Protein kinase assay Phosphorylation of MBPs (200 ng) by various protein kinases was carried out in a standard reaction mixture (10 µl) consisting of 40 mM Hepes-NaOH pH 8.0, 1 mM dithiothreitol, 0.1 mM EGTA, 5 mM Mg(CH3COO)2, 100 µM [γ-32P]ATP and an appropriate amount of kinase. The reactions were started by addition of the kinases, incubated at 30°C for 30 min, and stopped by addition of 10 µl of 2 × SDS-PAGE sample buffer. Phosphorylated proteins were subjected to SDS-PAGE and visualized by autoradiography. Phosphorylation of MBPs by CaMKIδ was carried out as described previously (19). In the case of ERK2, it was pre-activated by phosphorylation with MEK before the protein kinase assay. Briefly, ERK2 (500 ng) was incubated in a reaction mixture (20 µl) containing 40 mM Hepes-NaOH pH 8.0, 1 mM dithiothreitol, 0.1 mM EGTA, 5 mM Mg(CH3COO)2, 100 µM [γ-32P]ATP and MEK (50 ng). The reaction was started by addition of MEK, incubated at 30°C for 30 min, and stopped by placing the tube on ice. SDS-PAGE and western blotting SDS-PAGE was carried out essentially according to the method of Laemmli (20) in slab gels consisting of a 15% (w/v) acrylamide separating gel and a 3% (w/v) stacking gel. Western blotting was performed essentially as described previously (21). Other methods Protein concentrations were determined by the method of Bensadoun and Weinstein (22) using bovine serum albumin as a standard. Nucleotide sequences were determined by the dideoxynucleotide chain termination method with a BigDye Terminator Cycle Sequencing Ready Reaction Kit Ver.3.1 (Applied Biosystems) and a DNA Sequencer (model 3100; Applied Biosystems). Results Cloning of mouse MBP isoforms Commercial MBP isolated from bovine brain has been widely used as a substrate for protein kinase assays. Although the major component of natural MBP is the 18.5-kDa isoform, comparative analyses of other isoforms and recombinant MBPs have not been carried out yet. Therefore, in the first experiment, we attempted to prepare recombinant MBP isoforms using E. coli expression systems. We prepared cDNAs from mouse brain instead of bovine brain, and obtained three different clones for mouse MBP isoforms corresponding to 18.5-kDa MBP (isoform 5), 17-kDa MBP (isoform 6) and 14-kDa MBP (isoform 8). Mouse MBP(iso5) is the major constituent of the brain myelin sheath and shows the highest homology (90% identity in amino acid sequence) with bovine MBP. These three isoforms, MBP(iso5), MBP(iso6) and MBP(iso8), are known to be splice variants originating from the same gene (12, 13). Although the MBP isoforms are composed of different combinations of isoform-specific exons and common exons, their phosphorylation sites for various protein kinases are highly conserved (Fig. 1). Fig. 1 View largeDownload slide (A) Alignment of MBP isoforms. The amino acid sequences of bovine MBP and mouse MBP isoform 5 (Accession No.: P04370-5), isoform 6 (Accession No.: P04370-6) and isoform 8 (Accession No.: P04370-8) were aligned using CLUSTAL W. The arrowheads indicate putative phosphorylation sites. (B) Primary structures of MBP isoforms. The phosphorylation sites for PKA: A (3), protein kinase C: C (3), Ca2+/calmodulin-dependent protein kinase II: II (4), ERK2: E (5, 6) and Tyr kinase p56lck: L (7) are indicated by arrowheads. Fig. 1 View largeDownload slide (A) Alignment of MBP isoforms. The amino acid sequences of bovine MBP and mouse MBP isoform 5 (Accession No.: P04370-5), isoform 6 (Accession No.: P04370-6) and isoform 8 (Accession No.: P04370-8) were aligned using CLUSTAL W. The arrowheads indicate putative phosphorylation sites. (B) Primary structures of MBP isoforms. The phosphorylation sites for PKA: A (3), protein kinase C: C (3), Ca2+/calmodulin-dependent protein kinase II: II (4), ERK2: E (5, 6) and Tyr kinase p56lck: L (7) are indicated by arrowheads. Expression and purification of mouse MBP isoforms When the optimum conditions for expression of each mouse MBP isoform were employed, the recombinant MBPs were expressed efficiently using E. coli expression systems. The isoforms were expressed not only in the soluble fraction, but also as inclusion bodies in the insoluble fraction (Fig. 2A). Since the MBP isoforms were His6-tagged recombinant proteins, soluble fractions from the E. coli extracts were directly applied to a HiTrap Chelating HP column. Unexpectedly, however, all of the isoforms in the soluble fractions passed through the column (Fig. 2B). Since these MBP isoforms retaining a His6-tag at the C-terminal end passed through the affinity column, it is suggested that some inhibitory factors present in the crude samples prevented the recombinant MBPs from binding to the affinity column, as will be discussed later. Fig. 2 View largeDownload slide Expression of recombinant mouse MBP isoforms in E. coli. (A) Transfected E. coli cells were sonicated and separated into lysate (sup) and debris (ppt) fractions by centrifugation. Protein expression was analysed by SDS-PAGE followed by staining with Coomassie brilliant blue (left panel) and western blotting with an anti-His6 antibody (right panel). (B) Cell lysates from 200 ml of E. coli culture were applied to the HiTrap Chelating HP column. The applied samples (Lysate: L) and flow-through fractions (Pass: P) were resolved by SDS-PAGE and analysed by staining with Coomassie brilliant blue (left panel) and western blotting with an anti-His6 antibody (right panel). Fig. 2 View largeDownload slide Expression of recombinant mouse MBP isoforms in E. coli. (A) Transfected E. coli cells were sonicated and separated into lysate (sup) and debris (ppt) fractions by centrifugation. Protein expression was analysed by SDS-PAGE followed by staining with Coomassie brilliant blue (left panel) and western blotting with an anti-His6 antibody (right panel). (B) Cell lysates from 200 ml of E. coli culture were applied to the HiTrap Chelating HP column. The applied samples (Lysate: L) and flow-through fractions (Pass: P) were resolved by SDS-PAGE and analysed by staining with Coomassie brilliant blue (left panel) and western blotting with an anti-His6 antibody (right panel). We examined various conditions for purification of the recombinant MBPs using MBP(iso8) expressed in E. coli. Mouse MBP(iso8) in the soluble extract almost completely passed through the HiTrap Chelating HP column, indicating that direct affinity purification was not available (Fig. 3A). In previous papers, MBP was generally purified by several steps, including delipidation with organic solvent, extraction in acidic buffers and/or solubilization with urea (23–26). In the present study, we employed acid extraction with 0.2 M HCl as the first step of purification. By treatment with 0.2 M HCl, MBP could be extracted from both the soluble and insoluble fractions. With this treatment, most of the coexisting proteins were denatured and could be removed as insoluble precipitates. Consequently, MBP was efficiently extracted and recovered from E. coli lysates with 0.2 M HCl. After neutralization with NaOH, His6-tagged MBP was applied to HiTrap Chelating HP column. In this case, although MBP was completely absorbed onto the column, only a small amount of MBP was recovered from the column by elution with 200 mM imidazole (Fig. 3B). In the next experiment, recombinant MBP expressed in E. coli was first solubilized in 8 M urea and then applied to the HiTrap Chelating HP column. In this case, the recovery of MBP from the column was much improved, but the results were still insufficient (Fig. 3C). Next, we tried a combination of the acid extraction and urea treatment. The recombinant MBP expressed in E. coli was extracted with 0.2 M HCl, neutralized with 1 M NaOH, treated with 8 M urea, and then applied to the HiTrap Chelating HP column. With this procedure, we obtained the highest recovery of purified MBP after 200 mM imidazole elution from the HiTrap affinity column (Fig. 3D). Fig. 3 View largeDownload slide Purification of recombinant MBP(iso8) using the HiTrap Chelating HP column. (A) Cells expressing mouse MBP(iso8) were harvested from 200 ml of culture and lysed in buffer A. The resulting supernatant was applied to the HiTrap Chelating HP column. The column was washed and eluted with the indicated concentrations of imidazole. (B) The E. coli pellet was solubilized with 0.2 M HCl and MBP(iso8) was extracted. The acidic solution containing MBP(iso8) was neutralized, applied to the HiTrap Chelating HP column, and then washed and eluted with increasing concentrations of imidazole. (C) Escherichia coli cells expressing MBP(iso8) were solubilized in 8 M urea and applied to the HiTrap Chelating HP column. (D) The E. coli pellet was lysed in 0.2 M HCl and the extracted MBP(iso8) was neutralized and solubilized in 8 M urea. After the urea treatment, the solution was applied to the HiTrap Chelating HP column and eluted with imidazole as described above. The applied samples (sup) and eluted fractions were analysed by SDS-PAGE followed by staining with Coomassie brilliant blue. In the case of 0.2 M HCl treatment, the precipitate (ppt) after centrifugation of the acid extract was also analysed. Fig. 3 View largeDownload slide Purification of recombinant MBP(iso8) using the HiTrap Chelating HP column. (A) Cells expressing mouse MBP(iso8) were harvested from 200 ml of culture and lysed in buffer A. The resulting supernatant was applied to the HiTrap Chelating HP column. The column was washed and eluted with the indicated concentrations of imidazole. (B) The E. coli pellet was solubilized with 0.2 M HCl and MBP(iso8) was extracted. The acidic solution containing MBP(iso8) was neutralized, applied to the HiTrap Chelating HP column, and then washed and eluted with increasing concentrations of imidazole. (C) Escherichia coli cells expressing MBP(iso8) were solubilized in 8 M urea and applied to the HiTrap Chelating HP column. (D) The E. coli pellet was lysed in 0.2 M HCl and the extracted MBP(iso8) was neutralized and solubilized in 8 M urea. After the urea treatment, the solution was applied to the HiTrap Chelating HP column and eluted with imidazole as described above. The applied samples (sup) and eluted fractions were analysed by SDS-PAGE followed by staining with Coomassie brilliant blue. In the case of 0.2 M HCl treatment, the precipitate (ppt) after centrifugation of the acid extract was also analysed. We were able to purify all of the recombinant MBP isoforms with the aforementioned procedure with satisfactory recovery. In our experiments, approximately 16.0, 2.5 and 9.0 mg of purified MBP(iso5), MBP(iso6) and MBP(iso8), respectively, were obtained from 1 l of E. coli culture (Table I). Table I. Expression and purification of recombinant MBP from E. coli. Recombinant MBP  Escherichia coli strain  Induction (0.1 mM IPTG)   Purified MBP (mg)a  Time (h)  Temp (°C)  mMBP (iso 5)  RosettaI(DE3)  12  18  16.0  mMBP (iso 6)  BL21(DE3)  12  18  2.5  mMBP (iso 8)  RosettaII(DE3)  6  37  9.0  TandeMBP  BL21(DE3)  12  18  7.5  Recombinant MBP  Escherichia coli strain  Induction (0.1 mM IPTG)   Purified MBP (mg)a  Time (h)  Temp (°C)  mMBP (iso 5)  RosettaI(DE3)  12  18  16.0  mMBP (iso 6)  BL21(DE3)  12  18  2.5  mMBP (iso 8)  RosettaII(DE3)  6  37  9.0  TandeMBP  BL21(DE3)  12  18  7.5  aPurified preparation of recombinant MBP recovered from 1 l of E. coli culture. In vitro kinase assays using mouse MBP isoforms Protein kinase assays were carried out using the recombinant MBP isoforms as substrates and compared with natural bovine MBP. In these experiments, the MBP isoforms purified by the above-described method were phosphorylated by four different Ser/Thr protein kinases, namely CK1, DCLK, Dyrk1A and PKL01. Recombinant MBPs purified from E. coli were not phosphorylated without addition of the kinases, suggesting that purified preparations of MBP did not contain contaminating protein kinases in host bacteria (Fig. 4A, upper left panels). The mouse MBPs served as more efficient substrates than bovine MBP for all four protein kinases examined (Fig. 4A). Under the conditions used, the phosphate incorporation into MBP(iso6) was 8, 5, 1.6 and 2 times higher than that into bovine MBP when phosphorylated by CK1, DCLK, Dyrk1A and PKL01, respectively (Fig. 4B). Fig. 4 View largeDownload slide Phosphorylation of MBP isoforms by various Ser/Thr protein kinases. (A) Bovine MBP and mouse MBP isoforms were phosphorylated by CK1 (2.1 pmol), DCLK (1.6 pmol), Dyrk1A (1.2 pmol) or PKL01 (1.1 pmol) in a standard reaction mixture (10 µl) containing 100 µM [γ-32P]ATP as described in Materials and Methods. The reaction was performed at 30°C for 30 min and stopped by adding an equal volume of 2× SDS-PAGE sample buffer. The samples were subjected to SDS-PAGE and the phosphorylated proteins were detected by autoradiography (upper panels). The protein staining patterns with Coomassie brilliant blue are shown in the lower panels. (B) Quantification of phosphate incorporation into the MBPs. The radioactive bands shown in (A) were quantified using ImageJ software, and the rate of phosphorylation of each MBP isoform was calculated by setting the phosphorylation of bovine MBP as 1.0. Data are means ± SD values from three independent experiments. Fig. 4 View largeDownload slide Phosphorylation of MBP isoforms by various Ser/Thr protein kinases. (A) Bovine MBP and mouse MBP isoforms were phosphorylated by CK1 (2.1 pmol), DCLK (1.6 pmol), Dyrk1A (1.2 pmol) or PKL01 (1.1 pmol) in a standard reaction mixture (10 µl) containing 100 µM [γ-32P]ATP as described in Materials and Methods. The reaction was performed at 30°C for 30 min and stopped by adding an equal volume of 2× SDS-PAGE sample buffer. The samples were subjected to SDS-PAGE and the phosphorylated proteins were detected by autoradiography (upper panels). The protein staining patterns with Coomassie brilliant blue are shown in the lower panels. (B) Quantification of phosphate incorporation into the MBPs. The radioactive bands shown in (A) were quantified using ImageJ software, and the rate of phosphorylation of each MBP isoform was calculated by setting the phosphorylation of bovine MBP as 1.0. Data are means ± SD values from three independent experiments. Generation and purification of TandeMBP Purified preparations of the mouse MBP isoforms expressed in E. coli served as more efficient substrates for various protein kinases than commercially available bovine MBP. In an attempt to prepare an even more powerful substrate for protein kinase assays, we generated a unique recombinant protein substrate on the basis of mouse MBP. We produced a hybrid protein, in which two different mouse MBP isoforms, MBP(iso5) and MBP(iso6), were fused in tandem (Fig. 5A). MBP(iso5) was the most predominant MBP isoform and we were able to obtain it with the highest recovery (Table I). Among the MBP isoforms, MBP(iso6) served as the most efficient substrate for the various protein kinases examined. The hybrid protein consisting of MBP(iso5) and MBP(iso6) generated in this study was designated TandeMBP. TandeMBP showed considerable expression in E. coli, similar to the case for the other MBP isoforms, and was purified with the newly developed simple procedure (Fig. 5B). With this procedure, 7.5 mg of purified TandeMBP was obtained from 1 l of E. coli culture (Table I). Fig. 5 View largeDownload slide Purification of TandeMBP from E. coli. (A) Schematic illustration of the primary structure of TandeMBP. (B) Purification of TandeMBP from 600 ml of E. coli culture. The purification of TandeMBP was carried out using the same procedure used for MBP(iso8). The E. coli extract (−), supernatant (sup) and pellet (ppt) after 0.2 M HCl extraction, and eluted fractions from the affinity column were analysed by SDS-PAGE followed by staining with Coomassie brilliant blue. Fig. 5 View largeDownload slide Purification of TandeMBP from E. coli. (A) Schematic illustration of the primary structure of TandeMBP. (B) Purification of TandeMBP from 600 ml of E. coli culture. The purification of TandeMBP was carried out using the same procedure used for MBP(iso8). The E. coli extract (−), supernatant (sup) and pellet (ppt) after 0.2 M HCl extraction, and eluted fractions from the affinity column were analysed by SDS-PAGE followed by staining with Coomassie brilliant blue. Comparison of TandeMBP with the other MBP isoforms TandeMBP was phosphorylated by various Ser/Thr protein kinases, namely CK1, DCLK, CaMKIδ, Dyrk1A, ERK2, PKA and PKL01, and its phosphorylation levels were compared with those of the other MBP isoforms. As shown in Fig. 6A, TandeMBP served as a more efficient substrate for all the kinases examined in this study, when compared with bovine and mouse MBP isoforms. In particular, TandeMBP was 28 and 17 times more efficient as a substrate for CK1 and DCLK, respectively, than bovine MBP. For CaMKIδ, Dyrk1A, ERK2, PKA and PKL01, the phosphate incorporation into TandeMBP was 1.5−2.7-fold higher than that into bovine MBP (Fig. 6B). Fig. 6 View largeDownload slide Comparison of phosphorylation of TandeMBP with that of the other MBP isoforms. (A) Bovine MBP (b) and recombinant mouse MBP isoforms; MBP(iso5), MBP(iso6) and TandeMBP (T), were phosphorylated by CK1 (2.1 pmol), DCLK (1.6 pmol), Dyrk1A (1.2 pmol), ERK2 (2.4 pmol), PKL01 (1.1 pmol), PKA (0.2 pmol) or CaMKIδ (0.2 pmol) in a standard reaction mixture (10 µl) containing 100 µM [γ-32P]ATP as described in Materials and Methods. The reaction was performed at 30°C for 30 min and stopped by adding an equal volume of 2× SDS-PAGE sample buffer. The samples were subjected to SDS-PAGE, and the phosphorylated proteins were detected by autoradiography (upper panels). The protein staining patterns with Coomassie brilliant blue are shown in the lower panels. (B) Quantification of phosphate incorporation into the MBPs. The radioactive bands shown in (A) were quantified using ImageJ software and the rate of phosphorylation of each MBP isoform was calculated by setting the phosphorylation of bovine MBP as 1.0. Data are means ± SD values from three independent experiments. Fig. 6 View largeDownload slide Comparison of phosphorylation of TandeMBP with that of the other MBP isoforms. (A) Bovine MBP (b) and recombinant mouse MBP isoforms; MBP(iso5), MBP(iso6) and TandeMBP (T), were phosphorylated by CK1 (2.1 pmol), DCLK (1.6 pmol), Dyrk1A (1.2 pmol), ERK2 (2.4 pmol), PKL01 (1.1 pmol), PKA (0.2 pmol) or CaMKIδ (0.2 pmol) in a standard reaction mixture (10 µl) containing 100 µM [γ-32P]ATP as described in Materials and Methods. The reaction was performed at 30°C for 30 min and stopped by adding an equal volume of 2× SDS-PAGE sample buffer. The samples were subjected to SDS-PAGE, and the phosphorylated proteins were detected by autoradiography (upper panels). The protein staining patterns with Coomassie brilliant blue are shown in the lower panels. (B) Quantification of phosphate incorporation into the MBPs. The radioactive bands shown in (A) were quantified using ImageJ software and the rate of phosphorylation of each MBP isoform was calculated by setting the phosphorylation of bovine MBP as 1.0. Data are means ± SD values from three independent experiments. Discussion In the present study, we attempted to generate efficient substrates for various protein kinases that can replace commercial MBP isolated from bovine brain. First, we tried to obtain recombinant MBPs with a C-terminal His6-tag, because recombinant MBP was reported to be free from any post-translational modifications when expressed in E. coli (26). For this purpose, we obtained cDNA clones for the mouse MBP isoforms MBP(iso5), MBP(iso6) and MBP(iso8) corresponding to the 18.5-, 17- and 14-kDa MBP isoforms, respectively. Although we found the optimum conditions for expression of these isoforms in E. coli, the isoforms were expressed not only in the soluble fraction, but also in the insoluble fraction. When the supernatant fraction from each E. coli extract was applied to the HiTrap Chelating HP column, almost all of the MBP isoforms passed through the column even though they retained the His6-tag at their C-terminal end. The inability to purify the recombinant MBPs directly with the affinity column may have been caused by steric hindrance of each recombinant MBP arising from certain proteins binding to the C-terminal region. Therefore, we examined additional processes to remove any interacting proteins from the MBP isoforms before affinity purification with the HiTrap Chelating HP column. In this study, we found that the best protocol for purification of the recombinant MBP isoforms was as follows: each recombinant MBP was extracted from the E. coli lysate with 0.2 M HCl, neutralized, dissolved in 8 M urea, and then applied to the HiTrap Chelating HP column. In this protocol, both the acid extraction and the urea treatment were indispensable for efficient purification and the highest recovery of recombinant MBP from the affinity column. The urea treatment may prevent aggregation of MBP and also open out the structure of MBP to promote binding of His6-tagged MBP to the affinity column. By introducing this procedure, 16 mg of purified MBP(iso5) was obtained from 1 l of E. coli culture. Commercial MBP has frequently been used as an excellent substrate for various protein kinases. It was reported that intrinsic disorder in and around potential phosphorylation sites is an essential common feature for the target sites (11). Therefore, the reason why MBP serves as an efficient substrate for a variety of protein kinases may be attributed to its intrinsically disordered structure (8). When the phosphorylation level of the MBP isoforms by protein kinases were compared, MBP(iso6) served as the most efficient substrate among the MBP isoforms. MBP(iso8), the 14-kDa MBP isoform, is composed of the common exons Ib, III/IV, V and VII, while MBP(iso5) and MBP(iso6) contain exon VI and exon II, respectively, in addition to the common exons. The more efficient phosphorylation of MBP(iso6) by various protein kinases relative to the other isoforms may arise through the occurrence of additional phosphorylation sites in the extra 26-amino acid sequence generated by exon II. The recombinant MBP(iso5) was phosphorylated more efficiently than natural bovine MBP by the protein kinases examined, although the amino acid sequence and phosphorylation sites of these proteins are highly conserved. The possibility that bovine MBP we used was a pre-phosphorylated form was excluded by the fact that λphosphatase treatment of bovine MBP did not improve phosphorylation rate of this substrate (data not shown). Therefore, the difference in phosphorylation between the recombinant MBP and natural MBP could be explained by structural microheterogeneity arising from other post-translational modifications in bovine brain MBP or an additional C-terminal His6-tag sequence in the recombinant MBP. In the present study, we generated a novel and useful substrate based on the mouse MBP isoforms for in vitro protein kinase assays. This artificial substrate was composed of two MBP isoforms, namely MBP(iso5) on the N-terminal side and MBP(iso6) on the C-terminal side, which were connected in tandem. We designated this fusion MBP TandeMBP. TandeMBP was readily expressed in E. coli and purified with the same procedure developed for purification of the recombinant MBP isoforms. Furthermore, TandeMBP served as the most efficient substrate for all the Ser/Thr protein kinases examined to date. Interestingly, although TandeMBP was composed of MBP(iso5) and MBP(iso6), it served as a better substrate than the combined addition of these two isoforms. This may arise from specific conformation produced in TandeMBP, since the conformations of MBP(iso5) and MBP(iso6) in the fusion protein might be more accessible to protein kinases than those of the individual isoforms. TandeMBP generated in this study has many advantages over the commonly used MBP from bovine brain. We can obtain large amounts of the purified TandeMBP from E. coli expression systems without using bovine brain and can therefore avoid the risks of prion diseases. With the newly developed protocol, we can obtain this useful substrate for protein kinases at about one-hundredth lower cost than commercially available MBP. Furthermore, since TandeMBP serves as an excellent substrate for various Ser/Thr protein kinases, it will become a useful tool for in vitro assays to analyse the activities of many protein kinases. 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TI - TandeMBP: generation of a unique protein substrate for protein kinase assays
JF - The Journal of Biochemistry
DO - 10.1093/jb/mvu025
DA - 2014-04-08
UR - https://www.deepdyve.com/lp/oxford-university-press/tandembp-generation-of-a-unique-protein-substrate-for-protein-kinase-I4623qOiut
SP - 147
EP - 154
VL - 156
IS - 3
DP - DeepDyve
ER -