Recombinant production of self-assembling β-structured peptides using SUMO as a fusion partner

Abstract

Background: Self-assembling peptides that form nanostructured hydrogels are important biomaterials for tissue engineering scaffolds. The P₁₁-family of peptides includes, P₁₁-4 (QQRFEWEFEQQ) and the complementary peptides P₁₁-13 (EQEFEWEFEQE) and P₁₁-14 (QQOrnFOrnWOrnFOrnQQ). These form self-supporting hydrogels under physiological conditions (pH 7.4, 140 mM NaCl) either alone (P₁₁-4) or when mixed (P₁₁-13 and P₁₁-14). We report a SUMO-peptide expression strategy suitable for allowing release of native sequence peptide by SUMO protease cleavage.

Results: We have expressed SUMO-peptide fusion proteins from pET vectors by using autoinduction methods. Immobilised metal affinity chromatography was used to purify the fusion protein, followed by SUMO protease cleavage in water to release the peptides, which were recovered by reverse phase HPLC. The peptide samples were analysed by electrospray mass spectrometry and self-assembly was followed by circular dichroism and transmission electron microscopy.

Conclusions: The fusion proteins were produced in high yields and the β-structured peptides were efficiently released by SUMO protease resulting in peptides with no additional amino acid residues and with recoveries of 46% to 99%. The peptides behaved essentially the same as chemically synthesised and previously characterised recombinant peptides in self-assembly and biophysical assays.

Figure 1

Cloning strategy outlining the cloning of peptide coding regions at the BsaI restriction site of pET28_ SUMOadapt.

Figure 2

SDS-PAGE gels showing the cleavage of SUMO_P₁₁-N with SUMO protease. A) Uncleaved and SUMO protease cleaved SUMO_P₁₁-4 in either buffer (lanes 1 and 2) or water (lanes 3 and 4). B) SUMO_P₁₁-13 uncleaved (lane 1) and SUMO protease cleaved in water (lane 2) and C) SUMO_P₁₁-14 (K) uncleaved (lane 1) and SUMO protease cleaved in water (lane 2). Lanes 3 and 4 show overloaded samples of Lanes 1 and 2 respectively to allow visualisation the released P₁₁-14 (K) peptide (lane 4). Lane 5 shows SUMO protease.

Figure 3

Average absorbance traces of cleaved SUMO_P₁₁-N when purified using RP-HPLC on a C18 column. Absorbance measurements were made at 280 nm and 220 nm with the fraction collector programmed to collect peaks at 220 nm. The positions of the peaks subsequently identified to contain the peptide and SUMO protein are indicated by arrows. A) P₁₁-4 purification with the sharp peak at 6–7 minutes corresponding to P₁₁-4 and the broader peak between 11 and 20 minutes corresponding to SUMO. B) P₁₁-13 purification. C) P₁₁-14 (K) purification.

Figure 4

Hydrogel analysis of P₁₁-13/P₁₁-14. A) Hydrogel formed upon equimolar mixing of P₁₁-13 and P₁₁-14(K) indicated by arrow. B and C) Transmission electron microscopy (TEM) images of self-assembled B) P₁₁-4 at pH 2 and C) P₁₁-13/P₁₁-14(K). D) Circular dichroism analysis of P₁₁-4, P₁₁-13 and P₁₁-14(K) unimers, and P₁₁-4 and P₁₁-13/P₁₁-14(K) hydrogels at pH 7.4. Random coil conformation is observed for peptide unimers while a β-sheet conformation is observed for peptide hydrogels.