Expression and Purification of the VpDef Defensin in Escherichia coli using the Small Metal-Binding Proteins CusF3H+ and SmbP

Abstract

Background: The heterologous production of antimicrobial peptides in bacterial models can produce insoluble proteins due to the lack of proper folding. Fusion proteins have been used to increase the expression and solubility of these types of proteins with varying degrees of success. Objectives: Here, we demonstrate the use of the small metal-binding proteins CusF3H+ (9.9kDa) and SmbP (9.9kDa) as fusion partners for the soluble expression of the bioactive antimicrobial peptide VpDef(6.9 kDa) in Escherichia coli. Methods: The recombinant VpDef (rVpDef) peptide was expressed as a translational fusion with CusF3H+ and SmbP in Escherichia coli SHuffle under different small-scale culture conditions. The best conditions were applied to 1-liter cultures, with subsequent purification of the recombinant protein through IMAC chromatography. The recombinant protein was digested using enterokinase to liberate the peptide from the fusion protein, and a second IMAC chromatography step removed the fusion protein. The purified peptide was tested against two Gram-positive and two Gram-negative bacteria. Results: The use either of CusF3H+ or of SmbP results in recombinant proteins that are found in the soluble fraction of the bacterial lysate; these recombinant proteins are easily purified through IMAC chromatography, and rVpDef is readily separated following enterokinase treatment. The purified rVpDef peptide exhibits antimicrobial properties against both Gram-positive and Gram-negative. Conclusion: Use of the fusion proteins CusF3H+ and SmbP results in production of a soluble recombinant protein containing the antimicrobial peptide rVpDef that is correctly folded and that retains its antimicrobial properties once purified.

The Small Metal-Binding Protein SmbP Simplifies the Recombinant Expression and Purification of the Antimicrobial Peptide LL-37

“…A total of 3.6 mg of SmbP_LL-37 per liter of culture was obtained with a purity around 90% (as determined by ImageJ) with just one chromatographic step. The purity and amount of protein observed for SmbP_LL-37 were as expected based on the expression and purification of the SmbP-tagged Bin1B and VpDef peptides [5,6]. In the case of LL-37, although the amount of protein was relatively less, the purity was considerably higher.…”

“…We have previously reported the production of biologically active proteins, such as the human growth hormone, and the antimicrobial peptides (AMPs) Bin1b and VpDef tagged with SmbP, with satisfactory yields and purities [4][5][6]. Interestingly, in the case of the peptide Bin1b, we observed that the presence of SmbP, i.e., the complete SmbP_Bin1b protein construct, did not reduce the ability of the peptide to inhibit the growth of Staphylococcus aureus and Escherichia coli in its entirety.…”

High-Yield Expression and Purification of Scygonadin, an Antimicrobial Peptide, Using the Small Metal-Binding Protein SmbP

“…For example, we expressed the human growth hormone in E. coli BL21(DE3) using an engineered SmbP version containing the PelB signal sequence for protein translocation to the periplasm since disulfide bonds can be formed correctly on this site [24]. Alternatively, cationic antimicrobial peptides, like Bin1b, VpDef, and LL-37, have been expressed in the cytoplasm of E. coli SHuffle T7, where we confirmed the proper formation of disulfide bonds [25][26][27]. This specific strain of E. coli has mutations in the thioredoxin reductase (trxB) and glutathione reductase (gor) genes, which block the reductive pathways involving the enzymes allowing the formation of disulfide bonds.…”

“…The electrophoresis analysis showed the presence of SmbP_SCY in the flow-through; therefore, a larger column volume will retain more protein, increasing the total amount of protein. We can compare the amount of SmbP_SCY with the cationic antimicrobial peptides SmbP_Bin1b (4.4 mg/L), SmbP_VpDef (5.28 mg/L), and SmbP_LL-37 (3.6 mg/L) [ 25 , 26 , 27 ]. Our results showed we obtained almost ten times more protein than other antimicrobials produced; this difference might be because of the nature of each peptide [ 28 , 29 ].…”