Bacterial strains and plasmid constructions
E. coli DH5α was used as a host strain for the gene cloning and preparation of plasmids. E. coli BL21 (DE3) was used as a host strain for expression of the recombinant proteins. All E. coli strains are cultured in LB medium supplemented with antibiotics as needed.
Antimicrobial peptide magainin II (Mag II) [33], Metchnikowin (Metch) [35] and Andropin [34] were selected as interest peptides in this study. Mag II, Metch and Andropin gene were synthesized by overlap extension PCR according to their amino acid sequences, with a codon pattern adapted to the usage bias of E. coli [21, 43]. Fusion Mag II gene was first synthesized by overlap extension PCR with primer 1, primer 2, primer 3 and primer 4, then sequentially amplified with primer 8 and primer 4, primer 9 and primer 4, primer 10 and primer 4. The resulting amplicon encoded a fusion antimicrobial peptide Mag II with a sequence (ASRHWMAG) for the Ni(II)-dependent peptide bond hydrolysis [44], a His6-tag (HHHHHH) and a recognition site (ENLYFQ) by the site-specific protease TEV [45] at its N-terminus. The resulting sequence ASRHWMAGHHHHHHENLYFQ was denoted as NHT. Fusion Metch gene was also first synthesized by overlap extension PCR with primer 5, primer 6 and primer 7, and then sequentially amplified with primer 8 and primer 7, primer 9 and primer 7, primer 10 and primer 7. The resulting amplicon encoded a fusion peptide NHT-Metch. Similarly, fusion Andropin gene was first synthesized by overlap extension PCR with primer 11, primer 12, primer 13, primer 14 and primer 15, and then sequentially amplified with primer 8 and primer 15, primer 9 and primer 15, primer 10 and primer 15. The fusion Andropin gene was lastly constructed, encoding fusion peptide NHT-Andropin. Fusion antimicrobial peptides constructed were outlined in Fig. S1. The PagP gene (Gene ID: 946360) encoding its mature form was amplified using primer 16 and primer 17 from the chromosome of E. coli strain MG1655. The primers designed for gene amplification are listed in Supplementary data Table S1.
PagP is a Gram-negative bacterial outer membrane protein and extremely prone to accumulate in IBs when overexpressed in E. coli [2]. Base on the analysis of PagP sequence by web platform AGGRESCAN at http://bioinf.uab.es/aggrescan/ [27], seven aggregation “hot spots” (HSs) were identified, denoted as HS1, HS2, HS3, HS4, HS5, HS6 and HS7 successively. The C-terminal region of PagP (residue 101-161) is denoted as PagP-1, comprising four aggregation HSs (HS4, HS5, HS6 and HS7) (Fig. 3 and Fig. 4).
After being digested by EcoRI and HindIII, fusion Metch gene was inserted into expression vector pET30a and pET28b, generating vector pET30a-NHT-Metch and pET28b-NHT-Metch. The amplicon encoding mature PagP was digested with NdeI and BamHI, and then inserted into pET30a-NHT-Metch, generating vector pET30a-PagP-NHT-Metch for intracellular expression of the fusion protein PagP-NHT-Metch in E. coli. Similarly, after being digested by EcoRI and HindIII, fusion Mag II gene and Andropin gene were inserted into expression vector pET30a respectively, generating vector pET30a-NHT-Mag II and pET30a-NHT-Andropin. The amplicon encoding mature PagP was digested with NdeI and BamHI, and then inserted into pET30a-NHT-Mag II and pET30a-NHT-Andropin respectively, generating vector pET30a-PagP-NHT-Mag II and pET30a-PagP-NHT-Andropin for intracellular expression of the fusion protein PagP-NHT-Mag II and in PagP-NHT-Andropin in E. coli.
Using vector pET30a-PagP-NHT-Metch as template, fragment encoding fusion protein PagP-1-NHT-Metch was amplified with primer 18 and primer 7, and inserted into vector pET28b, generating expression vector pET28b-PagP-1-NHT-Metch. Similarly, corresponding fragments were amplified by using primer 19 and primer 7; primer 20, primer 21 and primer 7; primer 22 and primer 7; primer 23 and primer 7; primer 24 and primer 7, respectively. These fragments were inserted into vector pET28b respectively, generating vector pET28b-PagP-2-NHT-Metch, pET28b-PagP-3-NHT-Metch, pET28b-PagP-4-NHT-Metch, pET28b-PagP-5-NHT-Metch and pET28b-PagP-6-NHT-Metch. Furthermore, the fragment encoding NHT-Mag II was substituted for NHT-Metch gene, generating corresponding expression vector pET28b-PagP-1-NHT-Mag II, pET28b-PagP-2-NHT-Mag II and pET28b-PagP-3-NHT-Mag II. Similarly, the fragment encoding NHT-Andropin was substituted for NHT-Metch gene, generating corresponding expression vector pET28b-PagP-1-NHT-Andropin, pET28b-PagP-2-NHT-Andropin and pET28b-PagP-3-NHT-Andropin.
All constructs were checked by sequencing and primer sequences were listed in Supplementary data Table S1.
Site-specific mutation of PagP
Site-specific mutagenesis was performed to generate the mutants PagP on pET30a-PagP-NHT-Mag II background according to our previous report [46]. The resulting vectors are denoted as pET30a-P121L/P123L-PagP-NHT-Mag II, pET30a-P127I-PagP-NHT-Mag II, pET30a-P135L-PagP-NHT-Mag II, pET30a-P127I/P135L-PagP-NHT-Mag II, pET30a-P121L/P123L/P135L-PagP-NHT-Mag II, pET30a-P121L/P123L/P127I/P135L-PagP-NHT-Mag II, respectively. In the case of P121L/P123L-PagP, the residue Pro at position 121 and 123 was mutated to Leu with the primer 25 and primer 26. Similarly, mutant P127I-PagP was constructed with the primer 27 and primer 28, and P135L-PagP with primer 29 and primer 30. These mutants were also used as templates for further mutation. By performing the second round of mutation and the third round of mutation, the mutant P127I/P135L-PagP, P121L/P123L/P135L-PagP and P121L/P123L/P127I/P135L-PagP were constructed. All mutations were checked by sequencing.
Expression of fusion proteins
The E. coli cells (BL21 (DE3)) harboring the expression vectors were cultured overnight at 37°C in 6 mL of LB medium. The cultures were then diluted 100-fold and allowed to grow at 37°C until they reached the mid-log phase (with an optical density of around 0.6–0.8 at 600 nm). The expression of fusion proteins was induced by adding isopropyl β-D-1-thiogalactopy-ranoside (IPTG) to a final concentration of 0.3 mM. The resulting culture was further incubated for 12 h at 37°C. 100 mL of culture was centrifuged for 10 min at 6000 rpm to harvest bacteria at 4 °C. The E. coli cells were re-suspended in 10 mL lysis buffer (50 mM NaH2PO4-Na2HPO4, 0.2 M NaCl, 20 mM imidazole, pH 8.0), and then lysed by sonication on ice. After that, inclusion bodies were isolated by centrifugation for 15 min at 12000 rpm at 4°C, washed twice with washing buffer I (20 mM Tris-HCl, 50 mM NaCl, 0.1 % Triton X-100, 5 mM EDTA, pH 8.0), and then with washing buffer II (20 mM Tris-HCl, 50 mM NaCl, pH 8.0). IBs extracted or whole cell lysates were subjected to analysis by SDS-PAGE. The images of the gels were further analyzed using software (Image LabTM, BIO-RAD) to evaluate the expression levels of fusion proteins.
The specific hydrolysis of fusion proteins by Ni (II) ion
The specific hydrolysis of fusion proteins by Ni (II) ion was conducted under denatured condition according to the previous report [23]. The inclusion bodies isolated were dissolved in hydrolysis reaction buffer (20 mM Hepes, 6 M GuHCl, pH 8.2) with a concentration of about 200 μM. After that, fusion proteins were subjected to hydrolysis by addition of NiSO4 to final concentration of 5 mM, and incubated at 60°C for 12 h, 24 h or 36 h to investigate the hydrolysis process. The mixture was lastly diluted 5-fold with addition of lysis buffer (50 mM NaH2PO4-Na2HPO4, 0.3 M NaCl, 20 mM imidazole, pH 8.0), and centrifuged for 15 min at 12000 rpm at 4°C to remove the precipitated fusion tag.
Purification of recombinant antimicrobial peptides
After being specifically hydrolyzed by Ni2+, the fusion protein split into a tag and the NHT-AMP, which was further purified by Ni-chelating affinity chromatograph according to the protocol specified by the manufacturer (GE Healthcare Bio-Sciences). Firstly, the column was equilibrated with lysis buffer (50 mM NaH2PO4-Na2HPO4, 0.3 M NaCl, 20 mM imidazole, pH 8.0), and the protein sample was loaded. Then, the loaded column was washed three times with washing buffer III (50 mM NaH2PO4-Na2HPO4, 0.3 M NaCl, 40 mM imidazole, pH 8.0). After that, the recombinant NHT fusion (NHT-Metch, NHT-Mag or NHT-Andropin) was eluted with the elution buffer I (50 mM Na2HPO4-Na2HPO4, 200 mM imidazole, pH 7.0).
The fusion antimicrobial peptide (NHT-Metch, NHT-Mag or NHT-Andropin) contained in the elution buffer I was directly subjected to specific cleavage by addition of purified protease TEV at 25°C for 12 h according to the previous report [45]. The released AMP (Metch, Mag II or Andropin) was further purified using ion exchange chromatography (Macro-Prep® CM Resin, Bio-Rad). The column was first equilibrated with 2 column volumes (CV) of equilibration buffer (200mM imidazole, 50mM NaH2PO4-Na2HPO4, pH 7.0). 5 mL of the protein sample was loaded onto the column. Lastly, the recombinant AMP was eluted with elution buffer II (500 mM NaCl, 50mM Na2HPO4-Na2HPO4, pH 8.0).
Microbicidal activity assay of the recombinant antimicrobial peptides
The antimicrobial activities of the recombinant AMPs were analyzed by inhibition zone assay according to the protocol presented by previous report [47]. Briefly, gram-positive Staphylococcus aureus ATCC 25923 and gram-negative E. coli strain K12D31was grown overnight at 37°C in LB medium. 50 μL of these cultures were inoculated into 50 mL of fresh LB medium and incubated for additional 2-3 h at 37°C to OD600=0.5, respectively. 200 μL of cell suspension was inoculated into 50 mL of pre-warmed (45°C) LB medium containing 0.8 % (w/v) agar and rapidly dispersed. The medium was then poured into the Petri dish (9.0 cm in diameter) to form a uniform layer with a depth of about 1.5 mm. Holes with a diameter of 2 mm were punched into the gelated medium. For microbicidal activity assay, recombinant AMPs were added into the holes punched, and the plate was incubated at 37°C for 12 h to investigate appearance of the inhibition zones.