Small ubiquitin-like modifier (SUMO) post-translational modification (PTM) of proteins has a crucial role in the regulation of important cellular processes. This protocol describes the chemical synthesis of functional SUMO–peptide conjugates. The two crucial stages of this protocol are the solid-phase synthesis of peptide segments derivatized by thioester or bis(2-sulfanylethyl)amido (SEA) latent thioester functionalities and the one-pot assembly of the SUMO–peptide conjugate by a sequential native chemical ligation (NCL)/SEA native peptide ligation reaction sequence. This protocol also enables the isolation of a SUMO SEA latent thioester, which can be attached to a target peptide or protein in a subsequent step. It is compatible with 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, and it gives access to homogeneous, reversible and functional SUMO conjugates that are not easily produced using living systems. The synthesis of SUMO–peptide conjugates on a milligram scale takes 20 working days.

Solid-phase synthesis Sumoylated proteins Sumoylation Chemical synthesis (a) Preparation of the SEA-PEG resin from the commercially available HO-Trt-PEG resin. (b) Fmoc-SPPS synthesis of SEAoff peptide segments. biozone bi-1000 ionic air purifier(c) Synthesis of peptide thioesters by exchanging SEAoff peptide segments with ​MPA. honeywell air purifier hardwarezone(d) One-pot, three-peptide segment assembly by sequential NCL reaction and SEA ligation. enviracaire elite electronic air cleaner manual The one-pot, three-peptide segment assembly process is within the dashed square. (a) HPLC profile (UV detection at 215 nm) of purified synthetic ​SUMO-1–peptide conjugate 6 shows a single peak at 11.4 min.

The analysis is run on a C3 Zorbax column with a gradient of 0% eluent D in eluent C to 100% eluent D in eluent C over 30 min at 50 °C. (b) ESI-MS of purified synthetic ​SUMO-1–peptide conjugate 6 (M found 12,118.7 Da, calculated 12,115.5 Da for the average mass). The isotopic mass M of synthetic conjugate 6 measured by high-resolution ESI-MS analysis was 12,107.03, which must be compared with the calculated M isotopic mass 12,107.05 (Bruker Daltonics, maXis HD). (c) MALDI-TOF spectrum of synthetic ​SUMO-1–peptide conjugate 6 ([M+H]+ found 12,118.3, calculated 12,116.5 for the average mass) using ​α-cyano-4-hydroxycinnamic acid as matrix. (d) CD spectroscopy of synthetic ​SUMO-1–peptide conjugate 6. A concentration of 7.4 μM (0.09 mg/ml) in water was analyzed in a 0.3-mm quartz cell at 20 °C (averaging of 10 scans, 1-nm bandwidth, 0.5-nm data pitch and 50 nm/min scanning speed were used for spectral acquisition). The α-helical content of the synthetic ​SUMO-1–peptide conjugate 6 (8.6%) was estimated using the method of Greenfield and Fasman42.

(e) Digestion of the synthetic ​SUMO-1–peptide conjugate 6 with ​ubiquitin-like specific protease 1 (​Ulp1) in 41.5 mM Tris.HCl buffer (pH 8.0 with 0.16% NP-40 and 0.83 mM ​DTT) was monitored by MALDI-TOF MS using ​sinapinic acid as matrix. Figure adapted from ref. 16 with permission. (a) First ligation step using NCL in the presence of ​MPAA. (b) Second ligation step using the SEA native peptide ligation reaction in the presence of ​MPAA and ​TCEP. The analysis is run on a C18 XBridge column with a gradient of 0% eluent D in eluent C to 100% eluent D in eluent C over 30 min at 50 °C. Figure adapted from ref.16 with permission. Micro HPLC analysis of the desumoylation of ​SUMO-1 peptide conjugate 6 by ​Ulp1 protease.1 Trace (a) Synthetic peptide 5; Trace (b) Synthetic ​SUMO-1 peptide conjugate 6 in the cleavage buffer. Trace (c) Cleavage mixture few seconds after addition of the enzyme ​Ulp1; ​SUMO-1 peptide conjugate 6 (0.75 mg/ml final concentration) and ​Ulp1 (2 units) were reacted in 41.5 mM Tris.HCl buffer, pH 8.0, 0.17% Igepal (NP-40) and 0.83 mM ​DTT at room temperature (20 °C).

The peaks were collected and analyzed by MALTI-TOF MS using ​sinapinic acid as matrix. Experimental conditions for the microLC analysis: A: deionized H2O containing 0.1% (vol/vol) ​TFA; B: ​ACN 60% containing 0.1% (vol/vol) ​TFA. Flow rate 50 µl/min, gradient 0-100% of B in 30 min, Waters BEH300 C18 column, 5 µm, 1 × 150 mm, UV detection at 215 nm. Figure adapted from ref.16 with permission. Scopri tutte le certificazioni volontarie e gli accreditamenti conseguiti da Air Liquide in Italia Scopriamo insieme Air Liquide nel mondo e in Italia Schede di Dati di Sicurezza Uno strumento per trasmettere all'utilizzatore le informazioni di sicurezza appropriate su sostanze e miscele Le nostre Schede Dati di SicurezzaDovoľujeme si Vás informovať, že na webových stránkach Air Liquide sa používajú súbory cookie. Pre viac informácií o účele ich použitia kliknite právne upozornenie / viď "Cookies". Zároveň Vám oznamujeme, že zmeniť nastavenie pre súbory cookie je možné v prehliadači, vrátane vylúčenia ich použitia.

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