Additional file 1 of Calcium-assisted sortase A cleavage of SUMOylated metallothionein constructs leads to high-yield production of human MT3
Creators: name: Singh, Avinash Kumar
name: Krężel, Artur
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Biotechnology
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Figure S1. Sequences of fusion construct and purified WT-MT3. The WT-MT3 was expressed as a fusion protein with Chitin Binding Domain and Intein. The WT-MT3 (without any additional residues) was purified by IMPACT purification. Figure S2. Sequences of SUMO-MT3 fusion construct and purified GS-MT3. The GS-MT3 was expressed as a fusion protein with His6-SUMO. The fusion protein was purified by Ni2+-NTA and was then treated with Ulp1. The GS-MT3 (with extra Gly-Ser residues) was collected in flow through of the second Ni2+-NTA purification. Figure S3. Sequences of SUMO-LPETG-MT3 fusion construct and purified G-MT3. The LPETG-MT3 was expressed as a fusion protein with His6-SUMO. The fusion protein was purified by Ni2+-NTA and was then treated with eSrtA. The G-MT3 (with extra Gly residue) was collected in flow through of the second Ni2+-NTA purification. Figure S4. Sequences of eSrtA-MT3 fusion construct and purified G-MT3. This protein was expressed as a fusion protein having His6-SUMO-eSrtA-LPETG-MT3. The fusion protein was loaded on Ni2+-NTA resin and on-bead sortase-mediated auto-cleavage was induced by adding 5 mM CaCl2. The G-MT3 (with extra Gly residue) was collected in flow-through. Figure S5. Inducible on-bead sortase-mediated cleavage of eSrtA-MT3. A) SDS-PAGE profile of on-bead sortase-mediated cleavage of eSrtA-MT3. Lane 1 = protein ladder, Lane 2 = fusion protein after binding to the Ni2+-NTA beads, Lane 3 = cleavage of eSrtA-MT3 in the absence of Ca2+ ion, Lane 4 = cleavage of eSrtA-MT3 in the presence of Ca2+ ion, Lane 5 = purified G-MT3, Lane 6 = uncleaved eSrtA-MT3 left on the Ni2+-NTA beads, and lane 7 = protein ladder. B) Comparison of total protein yield (mg/L) obtained during Ca2+ induced on-bead cleavage of the eSrtA-MT3 in the presence and absence of external 10 µM eSrtA. Figure S56. SEC purification profile of apo-forms of WT-MT3, GS-MT3 (with additional Gly-Ser) and G-MT3 (with additional Gly) on a Superdex™ 75 Increase 10/300 GL column using 10 mM HCl, and absorbance was recorded at 220 and 280 nm. The retention volume for all apo-MTs was 19 mL. Figure S7. Deconvoluted ESI-MS spectra of WT-MT3, GS-MT3, G-MT3, and eSrtA obtained in this study. Figure S8. UV-vis monitored Zn(II) titrations to 1 µM metal-free G-MT3, GS-MT3, and MT3-WT in 50 mM borate pH 7.4, 100 mM NaClO4, 100 µM TCEP. Figure S9. SDS-PAGE purification profile of His6-eSrtA-LPETG-MT3 (eSrtA-MT3 without SUMO). Lane 1 = pellet, Lane 2 = lysate, Lane 3 = supernatant, Lane 4 = flowthrough, Lane 5 = wash, Lane 6 = eluted G-MT3, and lane 7 = protein ladder. A large fraction of the expressed protein was found in the pellet fraction. Loss of fusion protein during the washing step (with 50 mM imidazole) was also seen. Lane 8: A reference protein ladder (the same ladder as in lane 7). Table S1. Molecular masses and extinction coefficients of fusion constructs and purified proteins. Experimental masses were obtained from ESI-MS measurements. Table S2. List of all the primers used in this study.