Site-specific protein cleavage is essential for many protein-production protocols and typically requires proteases. We report the development of a chemical protein-cleavage method that is achieved through the use of a sequence-specific nickel-assisted cleavage (SNAC)-tag. We demonstrate that the SNAC-tag can be inserted before both water-soluble and membrane proteins to achieve fusion protein cleavage under biocompatible conditions with efficiency comparable to that of enzymes, and that the method works even when enzymatic cleavages fail.
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The data that support the findings of this study are available from the corresponding author upon reasonable request.
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We thank the Wells lab at UCSF for generously providing BirA enzymes, and A. Martinko and S. Pollock for helping us carry out phage biotinylation reactions. We thank the Craik lab at UCSF for providing the pCES1 phagemid vector, N. Sevillano for helping us build phage libraries, and M. Ravalin for help operating the multichannel peptide synthesizer. This work was supported in part by the National Institutes of Health (grant 5R35GM122603-02 to W.F.D.).
The authors declare no competing interests.
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Integrated supplementary information
Selection procedure (left) and proposed Ni2+ cleavage mechanism.
Cleavage conditions: peptide 0.2 mM, 0.1 M CHES, pH 8.2, 22 °C, 1 mM NiCl2 (black arrow indicates uncleaved peptide; ~55% cleavage yield).
SPI-2 cleavage comparison without glycine at the P1 position (left; 15% cleaved; * indicates uncleaved peptide) or with glycine at the P1 position (right; 90% cleaved; • indicates uncleaved peptide). Cleavage at pH 8.2, 0.1 M CHES, 1 mM NiCl2, 22 °C for 16 h. SPI-2 sequence without glycine at P1 position: EAAVCTTEWD PVCGKDGKTY SNLCWLNEAG VGLDHEGECL SRHWAPHHHH HH. SPI-2 sequence with glycine at P1 position: EAAVCTTEWD PVCGKDGKTY SNLCWLNEAG VGLDHEGECL GGSRHWGAPHHHH HH.
Supplementary Figure 4 Buffer and additive screening for cleaving peptide YFLGSRHWGGSRRLFY (0.2 mM).
Cleavage performed at pH 8.2, 1 mM NiCl2, 22 °C, for 18 h (left; buffer) or 6 h (right; additive). Other buffers screened—glycine, lysine, bicine, tricine—performed a lot worse. For additives, N-methyl hydroxylamine can speed up the cleavage, but the cleavage was not clean and N-methyl hydroxylamine was not adopted. N-hydroxyl piperidine, N,N-dimethyl hydroxylamine, diethanolamine, Boc-NHNH2, methoxylamine, hydroxylamine, and ethanolamine did not perform better than acetone oxime.
a1: YFLGGSHHTDLPGGSRRLFY-CONH2 Calc. 2,278.1 Da, obsd. 2,278.2 ± 0.2 Da. a2: YFLGG-COOH Calc. 555.3 Da, obsd. 555.3 ± 0.2 Da. a3: SHHTDLPGGSRRLFY-CONH2 Calc. 1,740.9 Da, obsd. 1,741.1 ± 0.2 Da. b1: YFLPGSRHWG-CONH2 Calc. 1,217.6 Da, obsd. 1,217.8 ± 0.2 Da. b2: YFLPG-COOH Calc. 595.3 Da, obsd. 595.3 ± 0.2 Da. b3: SRHWG-CONH2 Calc. 640.3 Da, obsd. 640.4 ± 0.2 Da. c1: YFLPGSHHWG-CONH2 Calc. 1,198.6 Da, obsd. 1,198.7 ± 0.2 Da. c2: YFLPG-COOH Calc. 595.3 Da, obsd. 595.3 ± 0.2 Da. c3: SHHWG-CONH2 Calc. 621.3 Da, obsd. 621.3 ± 0.2 Da.
Top, cleavage of His-tag-XXXXX-HB2225 constructs. Bottom, cleavage of His-tag-T4L-XXXXX-3hbtmV2 constructs. Lane 1 of each gel is prestained plus protein ladder.
His-tag-GSHHW-T4L (water-soluble protein; left) lane 1, t = 0; lane 2, cleavage at 18 h. His-tag-GSHHW-MBP (water-soluble protein; middle) lane 1, t = 0; lane 2, cleavage at 18 h. His-tag-T4L-GSHHW-NCHAMP1 (membrane protein; right) lane 1, t = 0; lane 2, cleavage at 4 h; lane 3, cleavage at 18 h. Cleavage conditions: 1 mM NiCl2, 0.1 M CHES, 0.1 M acetone oxime, 0.1 M NaCl, pH 8.2, protein 1 mg/mL, 22 °C (5 mM DPC added for His-tag-T4L-GSHHW-NCHAMP1). Full sequence of His-tag-GSHHW-T4L: MGSSHHHHHHSSGPGSHHWNIFEMLRIDEGLRLKIYKDTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNTNGVITKDEAEKLFNQDVDAAVRGILRNAKLKPVYDSLDAVRRAALINMVFQMGETGVAGFTNSLRMLQQKRWDEAAVNLAKSRWYNQTPNRAKRVITTFRTGTWDAYISRHWA. Full sequence of His-tag-GSHHW-MBP: MGSSHHHHHHSSGENLYFQGGNIGPGSHHWKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYAAGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDAALAAAQTNAAAISLHAA. Full sequence of His-tag-T4L-GSHHW-NCHAMP1: MGSSHHHHHHSSGLVPRGSHMGNIFEMLRIDEGLRLKIYKDTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNTNGVITKDEAEKLFNQDVDAAVRGILRNAKLKPVYDSLDAVRRAALINMVFQMGETGVAGFTNSLRMLQQKRWDEAAVNLAKSRWYNQTPNRAKRVITTFRTGTWDAYAAGGSGSTENLVPRGSKGNDYKDDDDKGGPGSHHWGGSGGSGGASDLDPLILTLXXXXXXXXXXXTVLALLSHRRTLQQK (de novo designed membrane protein, full sequence to be released in a separate paper).
Left, thrombin cleavage of T4L-LVPRGS-PL5. Cleavage conditions: protein 1 mg/mL, 50 mM Tris, pH 8.4, 200 mM NaCl, 0.5 mM CaCl2, 5 mM DPC, 22 °C, 16 h, thrombin: lane 2, 2 U/mL, lane 3, 6 U/mL, lane 4, 12 U/mL. One thrombin unit is defined as that amount of enzyme required to cleave 1 mg of a test protein when incubated in standard digest buffer at 20 °C for 16 h. Right, Ni2+ cleavage of T4L-GSHHW-PL5. Cleavage conditions: 1 mM NiCl2, 0.1 M CHES, 0.2 M NaCl, pH 8.2, 5 mM DPC, protein 1 mg/mL, 22 °C, 16 h. Full sequence of T4L-LVPRGS-PL5: MGSSHHHHHHSSGLHLRDAHMGNIFEMLRIDEGLRLKIYKDTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNTNGVITKDEAEKLFNQDVDAAVRGILRNAKLKPVYDSLDAVRRAALINMVFQMGETGVAGFTNSLRMLQQKRWDEAAVNLAKSRWYNQTPNRAKRVITTFRTGTWDAYAAGGSGSLVPRGSDPEQLKWISFCXXXXXXXXXXXXFMLYRGRR (de novo designed membrane protein, full sequence to be released in a separate paper). Full sequence of T4L-GSHHW-PL5: MGSSHHHHHHSSGLHLRDAHMGNIFEMLRIDEGLRLKIYKDTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNTNGVITKDEAEKLFNQDVDAAVRGILRNAKLKPVYDSLDAVRRAALINMVFQMGETGVAGFTNSLRMLQQKRWDEAAVNLAKSRWYNQTPNRAKRVITTFRTGTWDAYAAGGSGSGSHHWGSDPEQLKWISFCXXXXXXXXXXXXFMLYRGRR (de novo designed membrane protein, full sequence to be released in a separate paper).
His-tag-T4L-GSHHW-3hbtmV2 (left) and His-tag-GSHHW-HB2225 (right). Cleavage conditions: 1 mM NiCl2, 0.1 M CHES, 0.1 M acetone oxime, 0.1 M NaCl, pH 8.2, protein 1 mg/mL, 22 °C, 18 h, GuHCl or urea concentrations are as indicated in the figure, 5 mM DPC for His-tag-T4L-GSHHW-3hbtmV2.
Peptide 0.2 mM, 1 mM NiCl2, 0.1 M CHES, 0.1 M acetone oxime, pH 8.2, 22 °C. a. Cleavage reaction monitored at different time points. b. Mass measured by MALDI-TOF of uncleaved WCRLGSRHW-CONH2 (calc. 1,198.6 Da, obsd. 1,198.7 ± 0.2 Da) after 18 h, no obvious cysteine oxidation was observed. c. Mass measured by MALDI-TOF of cleaved peptide WCRL (calc. 633.3 Da, obsd. 633.3 ± 0.2 Da) after 18 h, no obvious cysteine oxidation was observed.
Cleavage at pH 8.2, 0.1 M CHES, 0.1 M acetone oxime, 0.1 M NaCl, 1 mM NiCl2, 5 mM DPC, 22 °C for 18 h. Left, supernatant after on-resin cleavage; right, eluted protein remained on Ni-NTA beads after cleavage.
Supplementary Figure 12 Cleavage of optimized peptide sequences in short peptide constructs and in protein constructs.
Left: HPLC trace is the cleavage of peptides YFLPGSHHWG and YFLHNSHHWG. Cleavage conditions: 1 mM NiCl2, 0.1 M CHES, pH 8.2, 22 °C, peptide 0.2 mM. Middle: lane 2, t = 0; cleavage of His-tag-PGSHHW-HB2225 (lane 3) and His-tag-HNSHHW-HB2225 (lane 4). Cleavage conditions: 1 mM NiCl2, 0.1 M CHES, 0.1 M acetone oxime, 0.1 M NaCl, pH 8.2, protein 1 mg/mL, 22 °C, 18 h. Right: lane 2, t = 0; cleavage of His-tag-T4L-PGSHHW-3hbtmV2 (lane 3), His-tag-T4L-HNSHHW-3hbtmV2 (lane 4). Cleavage conditions: 1 mM NiCl2, 0.1 M CHES, 0.1 M acetone oxime, 0.1 M NaCl, pH 8.2, protein 1 mg/mL, 22 °C, 18 h, 5 mM DPC.
Supplementary Figure 13 Cleavage time course for His-tag-T4L-GSHHW-3hbtmV2 (left) and His-tag-GSHHW-HB2225 (right).
Cleavage conditions: 1 mM NiCl2, 0.1 M CHES, 0.1 M acetone oxime, 0.1 M NaCl, pH 8.2, protein 1 mg/mL, 22 °C, 5 mM DPC for His-tag-T4L-GSHHW-3hbtmV2.
Cleavage conditions: 50 mM Tris, pH 8.0, 0.2 M NaCl, 1 mM DTT, 0.5 mM EDTA, 22 °C, 16 h, protein 2 mg/mL, TEV 0.02 mg/mL. Lane 1, before cleavage; lane 2, after cleavage.
Supplementary Figure 15 Peptide YFLGSRHWGGSRRLFY (0.2 mM) cleavage at different pH, 1 mM NiCl2, 22 °C, 0.1 M CHES.
pH 8.2, t = 2 h (13% cleavage); 5 h (40% cleavage). pH 8.6, t = 2 h (34% cleavage); 5 h (78% cleavage). pH 9.0, t = 2 h (71% cleavage); 5 h (97% cleavage).
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Dang, B., Mravic, M., Hu, H. et al. SNAC-tag for sequence-specific chemical protein cleavage. Nat Methods 16, 319–322 (2019). https://doi.org/10.1038/s41592-019-0357-3
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