Appl Microbiol Biotechnol DOI 10.1007/s00253-014-6077-9 BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Design and recombination expression of a novel plectasin-derived peptide MP1106 and its properties against Staphylococcus aureus Xintao Cao & Yong Zhang & Ruoyu Mao & Da Teng & Xiumin Wang & Jianhua Wang Received: 4 August 2014 /Revised: 5 September 2014 /Accepted: 7 September 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract A novel antimicrobial peptide MP1106 was de- hemolytic activity of only 1.16 % at a concentration of signed based on the parental peptide plectasin with four mu- 512 μg/ml and remained stable in human serum at 37 °C for tational sites and a high level of expression in Pichia pastoris 24 h. Furthermore, the activity of rMP1106 was minorly X-33 via the pPICZαA plasmid was achieved. The concen- affected by 10 mM dithiothreitol and 20 % dimethylsulfoxide. tration of total secreted protein in the fermented supernatant Our results indicate that MP1106 can be produced on a large was 2.134 g/l (29 °C), and the concentration of recombinant scale and has potential as a therapeutic drug against S. aureus. MP1106 (rMP1106) reached 1,808 mg/l after a 120-h induc- tion in a 5-l fermentor. The rMP1106 was purified using a Keywords MP1106 . Antimicrobial peptide . Pichia cation-exchange column, and the yield was 831 mg/l with pastoris . Stability . Staphylococcus aureus 94.68 % purity. The sample exhibited a narrow spectrum against some Gram-positive bacteria and strong antimicrobial activity against Staphylococcus aureus at low minimal inhib- Introduction itory concentrations (MICs) of 0.014, 1.8, 0.45, and 0.91 μM to S. aureus strains ATCC 25923, 29213, 6538, and 43300, The overuse of antibiotics has led to the emergence of respectively. Meanwhile, rMP1106 showed potent activity antibiotic-resistant pathogenic bacteria that has become a huge (0.03–1.8 μM) against 20 clinical isolates of methicillin- threat to public health. Many types of Staphylococcus aureus resistant S. aureus (MRSA). In addition, rMP1106 exhibited cause cutaneous and soft tissue infections (Miller and Cho a broad range of thermostability from 20 to 100 °C. The higher 2011). However, treating these infections is difficult because antimicrobial activity of rMP1106 was maintained in neutral of the emergence of multidrug-resistant strains (Lowy 1998). and alkaline environments (pH 6, 8, and 10), and its activity Methicillin was introduced into clinical use in 1960, and the was slightly reduced in acidic environments (pH 2 and 4). The first observations of methicillin-resistant S. aureus (MRSA) rMP1106 was resistant to the digestion of pepsin, snailase, and were reported in 1961 (Woodford and Livermore 2009). In proteinase K and was sensitive to trypsin. It exhibited recent years, infections caused by MRSA were detected in hospitals (hospital-acquired MRSA (HA-MRSA)), communi- Xintao Cao, Yong Zhang, and Ruoyu Mao contributed equally to this ties (community-acquired MRSA (CA-MRSA)), and live- paper. stock (livestock-acquired MRSA (LA-MRSA)) in a range of X. Cao : Y. Zhang : R. Mao : D. Teng : X. Wang : J. Wang animal species (Chambers and Deleo 2009;Kocketal.2011). Key Laboratory of Feed Biotechnology, Ministry of Agriculture, In addition, the CC398 strain of LA-MRSA, originally found Beijing 100081, China in pigs and cattle, was surprisingly detected in humans in 2003 : : : : : * (Graveland et al. 2011; van Cleef et al. 2011). Moreover, X. Cao Y. Zhang R. Mao D. Teng X. Wang J. Wang ( ) β Gene Engineering Laboratory, Feed Research Institute, Chinese MRSA strains are resistant to all members of the -lactam Academy of Agricultural Sciences, 12 Zhongguancun Nandajie St., class of antibiotics, including all penicillins, cephalosporins, Haidian District, Beijing 100081, People’sRepublicofChina and carbapenems, because they can produce penicillin- e-mail: [email protected] binding proteins (PBPs), known as PBP2 or PBP2a, that have β J. Wang low affinity to most -lactams and therefore confer resistance e-mail: [email protected] (Stefani et al. 2012; Woodford and Livermore 2009). Appl Microbiol Biotechnol Therefore, an urgent need exists for discovering and develop- Peptide design ing new antimicrobial drugs to control widespread MRSA. Antimicrobial peptides (AMPs) are potent pharmaceuticals The main structure of plectasin (α-helix from residues known for their rapid and broad-spectrum antimicrobial prop- 12–20 and β-sheets in residues 27–31 and 36–40) was erties, particularly those against antibiotic-resistant bacteria not changed to maintain or improve its expression level (Brogden and Brogden 2011). Plectasin, the first fungal in P. pastoris. Additionally, the amino acids containing a defensin with therapeutic potential that was especially targeted positive net charge and hydrophobic groups were intro- against Gram-positive bacteria, was identified from the sapro- duced to increase the antimicrobial activity of plectasin. phytic ascomycete Pseudoplectania nigrella (Mygind et al. As a result, S, K, and H residues containing hydroxyls 2005). This compound kills the pathogens by directly binding and a positive net charge were used to replace the polar Lipid II, the essential precursor of the cell wall (Schneider amino acid residues of D and Q at positions 9, 11, and et al. 2010). Additionally, plectasin showed no cytotoxicity to 14. Additionally, I, which contains a larger hydrophobic eukaryotic cells, a low induction of interleukin-8 (IL-8) pro- side chain, was used to replace the V residue in position duction (Hara et al. 2008), and no specific antibody responses 36. The amino acid sequences and properties of the in mice (Brinch et al. 2009). It also can be easily produced by parent peptides and designed peptide were calculated by prokaryotic and eukaryotic expression systems (Mygind et al. bioinformatics programs, including Antimicrobial Peptide 2005;Rothanetal.2013;Yangetal.2011; Zhang et al. 2011). Calculator and Predictor (http://aps.unmc.edu/AP/ The minimal inhibitory concentrations (MICs) of plectasin prediction/prediction_main.php), ProtParam (ExPASy against Streptococcus pneumonia and Staphylococcus Proteomics Server: http://www.expasy.org/tools/ epidermidis were 0.063–8and4–32 μg/ml, respectively. protparam.html), and Pepstats (EMBOSS biology However, the MICs of plectasin to S. aureus were 4– software: http://www.ebi.ac.uk/Tools/emboss/pepinfo/). 128 μg/ml (Hara et al. 2008;Mygindetal.2005), suggesting The mean hydrophobicity and relative hydrophobic plectasin activity needed to be increased to be effective against moment were calculated online using CCS scale (http:// S. aureus. www.bbcm.univ.trieste.it/wtossi/HydroCalc/HydroMCalc. The development of AMPs is still hampered by their tox- html). All of the parameters are summarized in Table 1. icity to mammalian cells and the lack of a cost-effective means The spatial structure of MP1106 was calculated by of commercial-scale production (Brogden 2005; Findlay et al. SWISS-Model (http://swissmodel.expasy.org/workspace// 2010; Brogden and Brogden 2011). A variety of approaches index.php?func=modelling_simple1&userid= have been employed to improve antimicrobial activity and USERID&token=TOKEN). reduce undesirable cytotoxic effects through optimization of natural AMP sequences (Chen et al. 2005; Jiang et al. 2011; Construction of the expression vector pPICZαA-MP1106 Lee et al. 2014). In this study, a variant of plectasin named MP1106 was designed and expressed in Pichia pastoris A 120-bp codon-optimized gene sequence of MP1106 was (named rMP1106). Additionally, the bactericidal activity, he- designed using the Reverse Translate Tool (www.bio- molytic activity, and stability of purified rMP1106 were informatics.org/sms2/rev_trans.html) based on the evaluated. preferential codon usage of P. pastoris (www.kazusa.or.jp/ codon/) (Yang et al. 2011). The DNA fragment contained the XhoI restriction site, a P. pastoris Kex2 protease Materials and methods cleavage site, the codon-optimized MP1106 open reading frame (ORF), two stop codons, and the XbaI restriction site Strains, plasmids, and reagents and was synthesized by Sangon Biotech (Shanghai, China). The DNA fragment and pPICZαA vectors were digested with Escherichia coli DH5α (Invitrogen, Beijing, China), XhoI and XbaI, gel-purified, and ligated together with T4 P. pastoris X-33 (Invitrogen, Beijing, China), and pPICZαA DNA ligase. The recombinant plasmid pPICZαA-MP1106 vectors (Invitrogen, Beijing, China) were used for cloning and was transformed into E. coli DH5α, and positive cells were expression. All of the strains used in antimicrobial assays and selected on zeocin-containing LB plates (25 μg/ml) and were their sources are summarized in Table 2. Vancomycin and screened by colony PCR with the following two pairs of ampicillin were purchased from the China Institute of Veter- primers: inary Drug Control. DNA restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (NEB, Primer 5′ AOX1: 5′-GACTGGTTCCAATTGACAAGC-3′ Beijing, China). The kits for plasmid extraction and DNA Primer 3′ AOX1: 5′-GCAAATGGCATTCTGACATCC-3′ purification were purchased from Tiangen (China). All other F1: 5′-CCGCTCGAGAAGAGAGGTTT-3′ chemical reagents used were of analytical grade. R1: 5′-GCTCTAGATTATTAGTAACAC-3′ Appl Microbiol Biotechnol Table 1 Sequences and key physicochemical parameters of Plectasin and MP1106 Peptides Sequence Mw pI GRAVYb μHrelc Charge Plectasin GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 4,401.99 7.77 −0.695 0.82 +2.0 MP1106 GFGCNGPWSEKDMHCHNHCKSIKGYKGGYCAKGGFICKCYa