Selenocysteine substitutions in thiyl radical enzymes

Abstract

Cysteine thiyl radicals are implicated as cofactors in a variety of enzymatic transformations, as well as transient byproducts of oxidative stress, yet their reactivity has undermined their detailed study. Selenocysteine exhibits a lower corresponding selenyl radical reduction potential, thus taming this radical reactivity without significant steric perturbation, potentially affording a glimpse into otherwise fleeting events in thiyl radical catalysis. In this chapter, we describe a suite of fusion protein constructs for general and efficient production of site-specifically incorporated selenoproteins by a recently developed nonsense suppression technology. As a proof of concept, we produced NikJ, a member of the radical S-adenosyl methionine enzyme family involved in the biosynthesis of peptidyl nucleoside antibiotics. We place emphasis throughout the plasmid assembly, protein expression, and selenium quantitation on accommodating the structural and functional diversity of thiyl radical enzymes. The protocol produces NikJ with near quantitative selenocysteine insertion, 50% nonsense read-through, and facile protein purification.

Keywords: Nonsense suppression; Radical SAM enzyme; Recombinant expression; Selenocysteine; Thiyl radical enzymes.

Fig. 1

Selenoprotein expression and purification strategy. (A) Schematic representation of pCm plasmids for selenoproteins expression and purification. Top: the common scaffold for all pCm plasmids with open variable region. Arrowed blocks represent coding sequences for: LacI, lactose operon repressor; H₈, octahistidine tag; CmR, chloramphenicol resistance gene. Bottom: the variable region of the pCm plasmids for fusion protein expression. SMT3, Saccharomyces cerevisiae SUMO homolog SMT3; MBP, maltose binding protein; TEV, tobacco etch Virus protease site; Ulp1, Ubl-specific protease 1; POI, protein of interest. A detailed view of the Ulp1 and TEV protease recognition sites is also provided. (B) User guide for selecting an appropriate plasmid for the expression and purification of a given selenocysteine mutant POI.

Fig. 2

Gibson assembly design for the cloning of a NikJ homolog into pCm family of plasmids. Top: schematic of the designed primers annealed with the Gibson assembly product DNA. Bold uppercase bases correspond to NikJ-coding DNA and the lowercase bases to the vector DNA, primers are shown as arrows. Bottom: list of primers designed for cloning a NikJ homolog into pCm1–6. To design custom primers for the cloning of a POI cDNA replace the bold bases for the corresponding cDNA of the POI gene.

Fig. 3

Expression and solubility of C₁₉₉U NikJ from pCm1–6 vectors. (A) His-tag stained SDS-PAGE gel of co-transformed BL21-AI E. coli cells with pSecUAG-Evol2 and pCm plasmids 1–6. Lanes were loaded with culture samples pre- and 24 h post-IPTG induction. Numbers above lanes corresponds to pCm vectors according to Fig. 1A. (B) His-tag stained SDS-PAGE of protein extraction from samples from induced cultures in A, lane numbering corresponds to plasmids numbers on A and “−” sign indicates insoluble cell debris fraction and “+” sign soluble protein fraction.

Fig. 4

Selenoprotein purification and protease cleavage. A SDS-PAGE analysis of NikJ-SMT3 C₁₉₉U purification. Lanes: 1, cell lysate debris; 2, cell lysate supernatant; 3, DNA precipitate; 4, DNA precipitation supernatant; 5, HisPur^™ Cobalt Resin column flow through at 20 mM imidazole; 6, wash with 20 mM imidazole, 7, eluted NikJ-SMT3 C₁₉₉U (400 mM imidazole); 8, 1:10 TEV digestion at time 0; 9, 1:10 TEV digestion at time 30 min; 10, TEV digested protein in the flow through of a HisPur^™ Cobalt Resin column; 11, eluted TEV and SMT3 (400 mM imidazole), 12, concentrated and desalted purified NikJ.

Fig. 5

SDS-PAGE analysis of protease cleavage of partially purified MBP-SMT3-NikJC₁₉₉U and NikJC₁₉₉U-SMT3 stained with Coomassie stain and also His-Tag stain. Lanes: 1, MBP-SMT3-NikJC₁₉₉U; 2, Ulp1 protease; 3, MBP-SMT3-NikJC₁₉₉U + ULP1 protease 0 min incubation; 4, MBP-SMT3-NikJC₁₉₉U + Ulp1 protease 30 min incubation; 5, TEV protease; 6, MBP-SMT3-NikJC₁₉₉U + TEV protease 0 min incubation; 7, MBP-SMT3-NikJC₁₉₉U + TEV protease 30 min incubation, 8, NikJC₁₉₉U-SMT3; 9, Ulp1 protease; 10, NikJC₁₉₉U-SMT3 + Ulp1 protease 0 min incubation; 11, NikJC₁₉₉U-SMT3 + Ulp1 protease 30 min incubation; 12, TEV protease; 13, NikJC₁₉₉U-SMT3 + TEV protease 0 min incubation; 14, NikJC₁₉₉U-SMT3 + TEV protease 30 min incubation.

Fig. 6

HPLC analysis of wild type and C₁₉₉U NikJ activity. NikJ assays were performed with 10 μM of NikJ in the presence of SAM (0.5 mM), EP-UMP (0.5 mM), sodium dithionite (2 mM), DTT (2 mM), and 40 mM NaCl in 50 mM HEPES pH 7.0 or 50 mM MES pH 6.0. The reactions were initiated by the addition of the enzyme and incubated at 28°C for 1320 min. At each time point, 15 μL of the assay mix was mixed with equal volume of ethanol and stored at −20°C. After removal of the protein precipitation by centrifugation, the supernatant was diluted fourfolds in 10 mM ammonium acetate pH 6.0, and analyzed by HPLC (Dionex IC5000+, Thermo) equipped with a DNAPac PA-100 column (Thermo). The HPLC analysis was performed with a linear gradient of 10–300 mM ammonium acetate pH 6.0 over 40 min.