A new strategy to produce a defensin: stable production of mutated NP-1 in nitrate reductase-deficient Chlorella ellipsoidea

Abstract

Defensins are small cationic peptides that could be used as the potential substitute for antibiotics. However, there is no efficient method for producing defensins. In this study, we developed a new strategy to produce defensin in nitrate reductase (NR)-deficient C. ellipsoidea (nrm-4). We constructed a plant expression vector carrying mutated NP-1 gene (mNP-1), a mature α-defensin NP-1 gene from rabbit with an additional initiator codon in the 5'-terminus, in which the selection markers were NptII and NR genes. We transferred mNP-1 into nrm-4 using electroporation and obtained many transgenic lines with high efficiency under selection chemicals G418 and NaNO(3). The mNP-1 was characterized using N-terminal sequencing after being isolated from transgenic lines. Excitingly, mNP-1 was produced at high levels (approximately 11.42 mg/l) even after 15 generations of continuous fermentation. In addition, mNP-1 had strong activity against Escherichia coli at 5 µg/ml. This research developed a new method for producing defensins using genetic engineering.

Figure 1. mNP-1 transformation and transgenic strain detection. (

A) A schematic map of the pGreen0029-NR-Ubi-mNP1-Nos plasmid. The pGreen0029-NR-Ubi-mNP1-Nos vector contained: an expression box of the NR gene from Chlorella ellipsoidea under the control of its own promoter (NR-P) and terminating in the 3′-UTR (NR-T) of the NR gene; an expression box of the mNP-1 gene (the mature NP-1 sequence with an additional Met at the N-terminus), controlled by the Ubiquitin gene-1 promoter (Ubi-P) and terminated by the Nos terminator (Nos-T); and an expression box of the neomycin phosphotransferase enzyme (NptII), which conferred resistance to aminoglycoside antibiotics. (B) PCR detection of the NptII gene using primers 1 and 2 (Table 1) and (C) identification of Ubi-mNP1-Nos fragments using primers 3 and 4 (Table 1) for transgenic nrm-4 cells. (1) pGreen0029-NR-Ubi-mNP1-Nos plasmid, (M) DNA molecular weight marker; (2) untransformed nrm-4 cells; (3–16) strains of transgenic nrm-4 cells.

Figure 2. Characterisation of the purified mNP-1.

(A) Tricine-SDS-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) of purified mNP-1. Extracted proteins from transgenic nrm-4 cells were dialysed with a molecular weight cut-off of 3,500 Da and chromatographed using a G-25 column (Pharmacia & Upjohn, NJ, USA). The fractions with the activity against E. coli were collected and continuously chromatographed 4 times. Purified peptides (15 µg per lane) from the G-25 chromatography were analysed using Tricine-SDS-PAGE (10–20% gel gradient). The mNP-1 migrates faster than a similarly sized molecular weight marker (M) due to its greater charge. mNP-1 denoted by 1 and 2. (B) Reversed-phase high-pressure liquid chromatography (RP-HPLC) of purified mNP-1. A mixture containing 20 µg each of mNP-1 was applied to a C-18 column and developed using a linear water-acetonitrile gradient at a flow rate of 1 ml/min and monitored at 280 nm on a UltiMate 3000 (Dionex, Sunnyvale, USA). (C) MALDI-TOF-MS analysis of purified mNP-1. The mNP-1 isolated from RP-HPLC was analysed by MALDI-TOF-MS using a Bruker Autoflex (Bruker, Germany). The peak at the molecular weight, 4023.3 Da, is the mNP-1 with a single charge; another peak of molecular weight 2013.7 Da is the mNP-1 containing two charges.

Figure 3. The detection of mNP-1 anti-microbial activity against E. coli. (

A) The MBC determination of Amp against E. coli ATCC25922. The 800 µg/ml Amp was used as initial concentration to detect the MBC against E. coli ATCC25922 by double multiple dilute methods using liquid LB medium. The concentration of bacteria incubated in this assay is 1 ×10⁶ CFU/ml. (1–6) 800, 400, 200, 100, 50 and 25 µg/ml Amp; (7) Negative control (without any Amp). (B) The MBC of mNP-1 against E. coli ATCC25922. An initial concentration of 80 µg/ml mNP-1 (obtained from RP-HPLC) was used to detect MBC against E. coli ATCC25922, and this solution was diluted multiple times by double-dilution methods using liquid LB medium. The concentration of bacteria used for this assay was 1 ×10⁶ CFU/ml. (1–6) 80, 40, 20, 10, 5 and 2.5 µg/ml mNP-1; (7) negative control (without any mNP-1). (C–E) Antimicrobial activity of total soluble protein extracted from transgenic nrm-4 cells. Total soluble protein extracted from cultures of transgenic nrm-4 cells without G418 selection stress (C), non-transgenic nrm-4 cells (D) and enlarged cultures of transgenic nrm-4 without G418 selection stress (E). (1) bacterial control; (2) negative control (LB liquid medium); (3–9) total soluble proteins diluted 2-, 4-, 8-, 16-, 32-, 64- and 128-fold with LB liquid medium, respectively.

Figure 4. Comparative analysis of mNP-1 and Amp against E. coli ATCC 25922.

E. coli ATCC 25922 (at a concentration of 1.4 ×10¹⁰ CFU/ml) was mixed with increasing concentrations of mNP-1 and Amp (concentration gradient: 0, 0.25, 0.5, 0.75, 1, 5, 10, 20, 40 and 50 µg/ml), respectively, and incubated at 37°C for 30 min. Surviving E. coli ATCC 25922 were counted using colony forming units (CFU) on plates. Results of bactericidal activity were represented as percentage survival versus concentration (i.e. CFU/ml) in the presence of mNP-1 and Amp. Upright figure is an enlarged part of Figure 4 at concentration of 0–1 µg/ml.