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. 2021 Oct 22;9:768854. doi: 10.3389/fchem.2021.768854

FIGURE 1.

FIGURE 1

Purification and activation of VcAEP. (A) Schematic representation of recombinant VcAEP constructs. Pro-VcAEP 1: full-length proenzyme form expressed with His-Ub-VcAEP (His = His6 tag, Ub = ubiquitin) and ending at P467. Pro-VcAEP 2: full-length VcAEP without the His-tag. Activated-VcAEP 3: active form of VcAEP after acidic autoactivation that begins with G36 and ends with N360. Truncated-VcAEP 4: truncated form of VcAEP, starting with Y62 and ending with D343. Cap 5: cleaved cap domain after acidic autoactivation, starting with A364 and ending with P467. The processing sites were determined by in-gel trypsin digestion followed by MS/MS de novo sequencing. NTR, N-terminal region; LR, linker region. (B) Purification, expression and activation of VcAEP. Left panel: analysis of purified fractions obtained from SEC chromatography, SDS-PAGE (upper) and western blot (lower). SDS-PAGE shows two bands, Pro-VcAEP 1 and Pro-VcAEP 2 (see panel A), with the lower band 2 being the VcAEP proenzyme without the His tag. Middle panel: SDS-PAGE analysis of the lauroylsarcosine-mediated acidic autoactivation of Pre-VcAEP 1 and Pro-VcAEP 2 and its products Activated-VcAEP 3, Truncated-VcAEP 4, and Cap 5, performed at pH 4–5 at 37°C for 30 min to generate Activated-VcAEP 3 (∼37 kDa). NA: no activation. Right panel: SDS-PAGE analysis of purified Activated-VcAEP 3 by SEC chromatography using pH 4 buffer.