Formation of active inclusion bodies induced by hydrophobic self-assembling peptide GFIL8.

Xu Wang,Bihong Zhou,Qing Zhao,Zhanglin Lin,Weike Hu

doi:10.1186/s12934-015-0270-0

Abstract

BackgroundIn the last few decades, several groups have observed that proteins expressed as inclusion bodies (IBs) in bacteria could still be biologically active when terminally fused to an appropriate aggregation-prone partner such as pyruvate oxidase from Paenibacillus polymyxa (PoxB). More recently, we have demonstrated that three amphipathic self-assembling peptides, an alpha helical peptide 18A, a beta-strand peptide ELK16, and a surfactant-like peptide L6KD, have properties that induce target proteins into active IBs. We have developed an efficient protein expression and purification approach for these active IBs by introducing a self-cleavable intein molecule.ResultsIn this study, the self-assembling peptide GFIL8 (GFILGFIL) with only hydrophobic residues was analyzed, and this peptide effectively induced the formation of cytoplasmic IBs in Escherichia coli when terminally attached to lipase A and amadoriase II. The protein aggregates in cells were confirmed by transmission electron microscopy analysis and retained ~50% of their specific activities relative to the native counterparts. We constructed an expression and separation coupled tag (ESCT) by incorporating an intein molecule, the Mxe GyrA intein. Soluble target proteins were successfully released from active IBs upon cleavage of the intein between the GFIL8 tag and the target protein, which was mediated by dithiothreitol. A variant of GFIL8, GFIL16 (GFILGFILGFILGFIL), improved the ESCT scheme by efficiently eliminating interference from the soluble intein-GFIL8 molecule. The yields of target proteins at the laboratory scale were 3.0–7.5 μg/mg wet cell pellet, which is comparable to the yields from similar ESCT constructs using 18A, ELK16, or the elastin-like peptide tag scheme.ConclusionsThe all-hydrophobic self-assembling peptide GFIL8 induced the formation of active IBs in E. coli when terminally attached to target proteins. GFIL8 and its variant GFIL16 can act as a “pull-down” tag to produce purified soluble proteins with reasonable quantity and purity from active aggregates. Owing to the structural simplicity, strong hydrophobicity, and high aggregating efficiency, these peptides can be further explored for enzyme production and immobilization.

Highlights

In the last few decades, several groups have observed that proteins expressed as inclusion bodies (IBs) in bacteria could still be biologically active when terminally fused to an appropriate aggregation-prone partner such as pyruvate oxidase from Paenibacillus polymyxa (PoxB)
In our previous studies [19, 20], we found three selfassembling amphipathic peptides were able to serve as “pull-down” fusion tags to effectively induce several normally soluble proteins into cytoplasmic active IBs in E. coli, i.e., an alpha-helical octadecapeptide 18A (EWLKAFYEKVLEKLKELF), a beta-strand peptide ELK16 (LELELKLKLELELKLK) [19], and a small surfactant-like peptide L6KD (LLLLLLKD) [20]
Hydrophobic peptide GFIL8 induced the formation of active IBs Unlike 18A, ELK16, and L6KD, the self-assembling tetrapeptide GFIL possesses neither polar nor electrically charged amino acids

Summary

Introduction

In the last few decades, several groups have observed that proteins expressed as inclusion bodies (IBs) in bacteria could still be biologically active when terminally fused to an appropriate aggregation-prone partner such as pyruvate oxidase from Paenibacillus polymyxa (PoxB). Several groups have observed that proteins deposited in IBs have biological activities. This was first reported by Worall in 1989 and 2 years later by Tokatlidis [6, 7]. The most universal and commonly used approaches to generate active IBs are to fuse a target protein to an aggregation-prone domain or protein sequence [8,9,10,11,12]. Several “pull-down” partners that drive proteins into active aggregates have

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Conclusion