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An engineered lantipeptide synthetase serves as a general leader peptide-dependent kinase.

Thibodeaux GN, van der Donk WA - Chem. Commun. (Camb.) (2012)

Bottom Line: Phosphorylation is an abundant post-translational modification involved in a myriad of cell signaling pathways.Herein, we have engineered the class II lantipeptide synthetase ProcM to generate a variety of peptides containing O-phosphoserine (pSer) and O-phosphothreonine (pThr) residues, either in vitro or in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, 600 S Mathews Ave, Urbana, IL 61801, USA.

ABSTRACT
Phosphorylation is an abundant post-translational modification involved in a myriad of cell signaling pathways. Herein, we have engineered the class II lantipeptide synthetase ProcM to generate a variety of peptides containing O-phosphoserine (pSer) and O-phosphothreonine (pThr) residues, either in vitro or in vivo.

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Mass spectra for (a) Proc-PKCμ and (b) Proc-H1 after removal of the leader peptide. Calculated and observed m/z values are shown in Table S1, ESI.†
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fig2: Mass spectra for (a) Proc-PKCμ and (b) Proc-H1 after removal of the leader peptide. Calculated and observed m/z values are shown in Table S1, ESI.†

Mentions: The methodology to prepare phosphorylated peptides in vitro is useful but difficult to scale up. Production of phosphopeptides by fermentation could potentially provide a solution. Prochlorosins have been successfully produced in E. coli by co-expression of ProcM and ProcA.21 Thus, the T516A and R510M ProcM mutants were co-expressed with ProcA2.8 in E. coli to evaluate their ability to produce phosphopeptides. Surprisingly, in these assays the ProcM-R510M mutant, which could not be purified in soluble form during the in vitro studies, clearly performed better than ProcM-T516A. Co-expression of the R510M mutant with the peptides Proc-PKCμ, Proc-H1 (histone H1 tail), Proc-CaM, and Proc-PKC (Table 1) resulted in phosphorylation of these peptides (Fig. 2 and Fig. S5 and S6a, ESI†). We then devoted some effort to larger scale production of a phosphorylated peptide. The crude mixture of phosphorylated and unphosphorylated His6-tagged Proc-PKC produced in E. coli was first purified by Ni-NTA affinity chromatography. The peptide was precipitated out of the imidazole-containing buffer and the ProcA 2.8 leader sequence was removed by proteolytic digestion with LysC. The digested peptide product was applied to a C18 HPLC column and the elution conditions were optimized to achieve base line resolution between phosphorylated and unphosphorylated PKC substrate (see ESI† and Fig. S6, ESI†). Thus, we were able to obtain highly pure, phosphorylated peptide from an in vivo biosynthesis reaction in a simple, two-step purification process. Comparable observations were made for two other peptides that were analyzed in a similar fashion (Proc-LR and Proc-PKCμ; Fig. S6, ESI†).


An engineered lantipeptide synthetase serves as a general leader peptide-dependent kinase.

Thibodeaux GN, van der Donk WA - Chem. Commun. (Camb.) (2012)

Mass spectra for (a) Proc-PKCμ and (b) Proc-H1 after removal of the leader peptide. Calculated and observed m/z values are shown in Table S1, ESI.†
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475617&req=5

fig2: Mass spectra for (a) Proc-PKCμ and (b) Proc-H1 after removal of the leader peptide. Calculated and observed m/z values are shown in Table S1, ESI.†
Mentions: The methodology to prepare phosphorylated peptides in vitro is useful but difficult to scale up. Production of phosphopeptides by fermentation could potentially provide a solution. Prochlorosins have been successfully produced in E. coli by co-expression of ProcM and ProcA.21 Thus, the T516A and R510M ProcM mutants were co-expressed with ProcA2.8 in E. coli to evaluate their ability to produce phosphopeptides. Surprisingly, in these assays the ProcM-R510M mutant, which could not be purified in soluble form during the in vitro studies, clearly performed better than ProcM-T516A. Co-expression of the R510M mutant with the peptides Proc-PKCμ, Proc-H1 (histone H1 tail), Proc-CaM, and Proc-PKC (Table 1) resulted in phosphorylation of these peptides (Fig. 2 and Fig. S5 and S6a, ESI†). We then devoted some effort to larger scale production of a phosphorylated peptide. The crude mixture of phosphorylated and unphosphorylated His6-tagged Proc-PKC produced in E. coli was first purified by Ni-NTA affinity chromatography. The peptide was precipitated out of the imidazole-containing buffer and the ProcA 2.8 leader sequence was removed by proteolytic digestion with LysC. The digested peptide product was applied to a C18 HPLC column and the elution conditions were optimized to achieve base line resolution between phosphorylated and unphosphorylated PKC substrate (see ESI† and Fig. S6, ESI†). Thus, we were able to obtain highly pure, phosphorylated peptide from an in vivo biosynthesis reaction in a simple, two-step purification process. Comparable observations were made for two other peptides that were analyzed in a similar fashion (Proc-LR and Proc-PKCμ; Fig. S6, ESI†).

Bottom Line: Phosphorylation is an abundant post-translational modification involved in a myriad of cell signaling pathways.Herein, we have engineered the class II lantipeptide synthetase ProcM to generate a variety of peptides containing O-phosphoserine (pSer) and O-phosphothreonine (pThr) residues, either in vitro or in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, 600 S Mathews Ave, Urbana, IL 61801, USA.

ABSTRACT
Phosphorylation is an abundant post-translational modification involved in a myriad of cell signaling pathways. Herein, we have engineered the class II lantipeptide synthetase ProcM to generate a variety of peptides containing O-phosphoserine (pSer) and O-phosphothreonine (pThr) residues, either in vitro or in vivo.

Show MeSH