Expression and purification of an antitumor‐analgesic peptide from the venom of Mesobuthus martensii Karsch by small ubiquitin–related modifier fusion in Escherichia coli

Abstract

To prevent protein aggregation, some proteins are usually expressed as fusion proteins from which target proteins can be released by proteolytic or chemical reagents. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the antitumor-analgesic peptide from the venom of Buthus martensii (Karsch) scorpion (AGAP). The optimal expression level of the soluble fusion protein, SUMO-AGAP, was up to 40% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease (Ulp1) to obtain the recombinant AGAP (rAGAP), which was further purified by Ni-NTA affinity chromatography. The purified final product was >95% pure by SDS-PAGE stained with Coomassie brilliant blue R-250. Mass spectroscopic analysis indicated the protein to be 7142.63 Dalton, which equaled the theoretically expected mass. N-terminal sequencing of rAGAP showed the sequence corresponded to the native protein. MTT assay indicated the rAGAP could significantly inhibit the proliferation of Jurkat and Hut 78 T lymphoma cell lines. The further writhing experiment showed that the rAGAP had an intensive analgesic effect. The expression strategy presented in this study allows convenient high yield and easy purification of the rAGAP with native sequences.

In vitro refolding and functional analysis of polyhistidine-tagged Buthus martensii Karsch antitumor-analgesic peptide produced in Escherichia coli

“…Similar to other small peptides rich in disulfide bridges, high level expression of rAGAP in Escherichia coli is still a technical difficulty to produce a reduced cytosolic environment (Baneyx and Mujacic 2004). Although several approaches have been used to promote soluble expression of AGAP in Escherichia coli, and SUMO fusion technology has dramatically improved expression of soluble rAGAP, unsatisfactory yields and bioactivities remain a major bottleneck for further application (Cao et al 2010;Liu et al 2003). Promotion of disulfide bridge formation in Escherichia coli and in vitro oxidative renaturation are still good choices for production of high active AGAP.…”

“…Most scorpion analgesic peptides, but not BmK AGAP, are b-toxins (Ma et al 2010) and act on voltage-sensitive sodium channels. BmK AGAP exhibits analgesic activity and antitumor activity in vivo (Cao et al 2010;Liu et al 2003) and inhibits the proliferation and migration of many malignant tumor cell lines.…”

Analgesic-antitumor peptide inhibits the migration and invasion of HepG2 cells by an upregulated VGSC β1 subunit

“…Moreover, these polypeptides have been shown to affect the activities and functions of ion channels, such as sodium and potassium channels [2,3]. Analgesic-antitumor peptide (AGAP), one of these polypeptides, has been purified from the venom of B. martensii Karsch, and several studies have shown that AGAP has analgesic and antitumor activities [4,5]. For example, AGAP markedly prolongs the survival of mice with engrafted Ehrlich ascites tumor cells and suppresses the growth of S-180 fibrosarcoma efficiently.…”

Analgesic-antitumor peptide induces apoptosis and inhibits the proliferation of SW480 human colon cancer cells

“…Various toxic polypeptides, usually called scorpion toxins, were considered to be the major bioactive ingredients of OAS. Recently, one of these polypeptides, analgesic-antitumor peptide (AGAP), has been purified and its analgesic and antitumor activities have also been demonstrated in pharmacological studies (4,5). Small ubiquitin-related modifier-AGAP (SUMO-AGAP) is a product of recombinant AGAP (rAGAP) linked with a hexa-histidine tag by Escherichia coli ( E. coli ).…”

“…Small ubiquitin-related modifier-AGAP (SUMO-AGAP) is a product of recombinant AGAP (rAGAP) linked with a hexa-histidine tag by Escherichia coli ( E. coli ). In this expression system, SUMO was capable of significantly enhancing the expression and efficiently improving the accurate folding of AGAP; thereby, rAGAP significantly inhibited the proliferation of lymphoma and glioma (5). Accordingly, rAGAP appears to be an ideal candidate drug for antitumor therapy.…”