A novel anti-lipopolysaccharide factor from blue swimmer crab Portunus pelagicus and its cytotoxic effect on the prokaryotic expression host, E. coli on heterologous expression
Journal of Genetic Engineering and Biotechnology volume 21, Article number: 22 (2023)
Invertebrates like crabs employ their own immune systems to fight against a number of invasive infections. Anti-lipopolysaccharide factors (ALFs) are an important class of antimicrobial peptides (AMPs) exhibiting binding and neutralizing activities against lipopolysaccharides.
This study identified and characterized a novel homolog of ALF (Pp-ALF) from the blue swimmer crab Portunus pelagicus. Pp-ALF has a 369bp open-reading frame encoding a protein with 123 amino acids. The deduced protein featured an LPS-binding domain and a signal peptide. The predicted tertiary structure of Pp-ALF contains three α helices packed against four β sheets. The deduced amino acid sequence of Pp-ALF had a net positive charge of +10.75 and an isoelectric point of 9.8. Phylogenetic analysis revealed that Pp-ALF has a strong ancestral relationship with crab ALFs.
Antibacterial, antiviral, antifungal, anticancer, and antibiofilm activities of Pp-ALF could be revealed by in silico prediction tools. Recombinant expression of Pp-ALF was unsuccessful in the Escherichia coli Rosetta-gami expression system due to the cytotoxic effect of the peptide to the host. The toxic effect of Pp-ALF to the host was displayed by membrane permeabilization and death of the host cells by fluorescent staining with Syto9-Propidium Iodide and CTC-DAPI- FITC.
Emerging issues with antibiotic resistance in microbial pathogens have become a serious threat to the health sector . It is difficult to prevent antibiotic resistance, since it results from adaptation, besides being a fundamental aspect of microbial evolution . Therefore, novel molecules are required as alternatives to antibiotics. Since AMPs operate concurrently on various target sites, they are less likely to cause resistance development compared to antibiotics. They are typically short cationic molecules that are found throughout the entire living kingdom and have a wide range of antibacterial, antifungal, antiviral, and antiparasitic actions .
In crustaceans, anti-lipopolysaccharide factors (ALFs) are considered as important antimicrobial peptides that bind to lipopolysaccharides inhibiting the microorganisms . Furthermore, ALFs mediate hemoglobin granulation and the intracellular coagulation cascade [5, 6]. The first ALF was discovered in horse shoe crab, Limulus polyphemus . Numerous ALF homologs have now been described in a wide variety of crustacean taxa, including shrimps [8, 4, 9, 10], cray fishes , lobsters , and crabs [13, 14]. Different isoforms of ALFs have been found in crustacean species; there is very little overlap between the same or related species . Eleven isoforms of ALFs have been identified in Penaeus monodon with antibacterial, antifungal, and antiviral functions .
Technology for the production of AMPs is developing quickly. The extraction of natural resources, chemical synthesis, and recombinant expression systems are the three important technologies . Among these, heterologous expression is considered better due to its affordability and effectiveness.
Blue swimming crab or flower crab, Portunus pelagicus is an economically important species in tropical and subtropical oceans [18, 19]. Typically, the shallow bays with sandy bottoms are home to huge populations of this species . Crustaceans lack complex and highly specific adaptive immune system like vertebrates and their defense mainly rely on innate immune responses . AMPs play an important role in the innate defense mechanism of crabs like crustaceans. So, the identification and characterization of novel AMPs from P. pelagicus would be useful for health management in crustacean culture systems.
In the present study, ALF from P. pelagicus was cloned and characterized. Even though the recombinant expression of P. pelagicus ALF was tried in Escherichia coli, the peptide was found to be lethal to the prokaryotic host E. coli. A eukaryotic expression system would be a probable solution to this problem.
Experimental organism and hemolymph collection
Live and healthy Portunus pelagicus collected from the backwaters of Cochin, Kerala, India, were used for the study. Hemolymph was collected from the base of abdominal appendages using specifically designed capillary tubes (RNase-free) rinsed using pre-cooled anticoagulant −10% sodium citrate, pH 7.0. The collected hemolymph was transferred into TRI reagent (Sigma-Aldrich) and stored at −80°C.
Total RNA isolation and reverse transcription
The total RNA was isolated from P. pelagicus hemolymph using TRI reagent followed by the manufacturer’s protocol. The quantity and quality of the isolated total RNA were checked spectrophotometrically (A260:A280) and 0.8% agarose gel electrophoresis, respectively. The first-strand cDNA was generated in a total of 20μL reaction mixture with 5μg total RNA, 1X RT buffer, 2mM dNTPs, 2μM oligo d (T20), 20U of RNase inhibitor, and 100U of M-MLV Reverse transcriptase. The reaction was carried out for 1h at 42°C followed by an inactivation step at 85°C for 15 min. To check the quality of RNA, an internal control gene β actin (forward- 5′ CTTGTGGTTGACAATGGCTCCG-3′; Reverse- 5′ TGGTGAAGGAGTAGCCACGCTC-3′) was used.
PCR amplification and TA cloning
Amplification of ALF sequence from cDNA of P. pelagicus was done using ALF primers (Forward- 5' AGGGAGTGGGTGATGAGCTA 3'; Reverse- 5' TACGGCTATTACGATCCAACA 3'). The reaction volume for PCR was setup by 25μL with 1X standard Taq buffer (10 mMTris-HCl, 50 mMKCl, pH 8.3), 3.5 mM MgCl2, 200 μM dNTPs, 0.4 μM each primer, and 1 U TaqDNA polymerase (New England Biolabs, USA). The thermal profile for the reaction was as follows; initial denaturation at 95 °C for 2 min followed by 35 cycles of 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s and a final extension at 72 °C for 10 min. Visualization of the amplicons was done by 1.5% agarose gel electrophoresis stained with ethidium bromide using Syngene G: Box Gel documentation unit. The PCR product was purified and cloned in to pGEM®-T Easy cloning vector (Promega). The cloned product was transformed into competent DH5α E. coli cells, as per the manufacturer’s protocol. Positive clones were isolated by blue/white screening and confirmed by PCR with gene-specific and vector-specific primers (5′ TGTAATACGA CTCACTATAGGG 3′; Sp6 R (5′ GATTTAGGTGACACTATAG 3′). From the positive clones, single white colony was selected for plasmid isolation. Plasmid DNA was extracted using GenElute™ HP Plasmid Miniprep Kit (Sigma). The isolated plasmid was sequenced with vector-specific primers on an ABI Prism 377 DNA sequencer (Applied Biosystem) at Agrigenome Sequencing Facility, India.
Molecular characterization of Pp-ALF in silico
The nucleotide sequences were assembled and converted into respective amino acid sequences by Expert Protein Analysis System (ExPASy) (http://web.expasy.org/translate/). BLASTn and BLASTp of the National Centre for Biotechnology Information (NCBI) were used for homology searches of nucleotide sequences and deduced amino acids, respectively. The processing site for mature peptide and signal peptide cleavage site were predicted using SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP-3.0/). The physico-chemical characterization of the mature peptide was performed with Protparam tool (http://cn.expasy.org/cgi-bin/protparam). Kyte& Doolittle plot in ProtScale tool of ExPASy ((https://web.expasy.org/protscale) was used to analyze the hydrophobicity over the length of the peptide sequence. Amphipathicity and hydrophobic phase of the lipopolysaccharide domain was elucidated by Heliquest online tool (http://heliquest.ipmc.cnrs.fr/cgibin/ComputParamsV2.py).
Multiple sequence alignment and phylogenetic analysis
Multiple sequence alignment was used for finding conserved domains and motifs of P. pelagicus ALF with other ALF sequences retrieved from GenBank. The sequences were aligned by the ClustalW algorithm of BioEdit. The maximum likelihood phylogenetic tree of selected sequences retrieved from NCBI GenBank was constructed with MEGA 7 software based on amino acid sequences. The reliability of the branches was tested using bootstrap resampling with 1000 pseudo-replicates.
The RNA sequence (http://www.fr33.net/seqedit.php) of Pp-ALF was extracted from the corresponding cDNA sequence and submitted to the RNA structure web server (http://ac.at/cgi-bin/RNAfold.cgi). RNA structure with minimum free energy was predicted by this tool. The secondary structure of the primary amino acid sequence of P. pelagicus ALF was elucidated with PSIPRED server ((http://bioinf.cs.ucl.ac.uk/psipred). The spatial structure was constructed by Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) algorithm based on homology model templates and viewed by pyMOL software. The Ramachandran plot was constructed by SAVESv6.0 - Structure Validation Server (https://saves.mbi.ucla.edu) to assess the stereochemical quality of the putative protein structure
Functional analysis in silico
The antimicrobial peptide database (APD3) (http://aps.unmc.edu/AP/main.php) predicted the Wimley-White whole-residue hydrophobicity and Boman index of the peptide. CAMPR3 (www.camp3.bicnirrh.res.in), a database of antimicrobial peptide sequences, structures, and signatures, performed sequence optimization prediction with respect to the antibacterial activity of the peptide. The peptide, which is prone to aggregation, was examined with AGGRESCAN (http://bioinf.uab.es/aggrescan/) to determine the short and particular regions of active residues. The tendency of the functional kinds of AMPs was predicted using iAMPpred (http://cabgrid.res.in:8080/amppred/index.html). Anticp (webs.iiitd.edu.in/raghava/anticp/) server computed the probability of the peptide to be an anticancer peptide and antiangiopred (http://crdd.osdd.net/raghava/antiangiopred/) predicted the angiogenesis inhibition property of the peptide. The immunomedicine group (http://imed.med.ucm.es/Tools/antigenic.html) predicted the antigenic property of the peptide. Anti-inflammatory and anti-tubercular properties of the peptide were predicted by AIPpred (http://www.thegleelab.org/AIPpred/) and AntiTbpred (http://thegleelab.org/AtbPpred), respectively. The dPABBs webserver (http://ab-openlab.csir.res.in/abp/antibiofilm/) predicted biofilm inhibitory properties of the peptide. The hemolytic activity of Pp-ALF was analyzed by HemoPred (http://codes.bio/hemopred/) and cell-penetrating ability of the peptide was checked by CellPPD server (http://crdd.osdd.net/raghava/cellppd/). DNA-protein interaction-based prediction was evaluated by DNAbinder (http://crdd.osdd.net/raghava/dnabinder/). The immunomodulatory property of peptides was examined by VaxinPAD (https://webs.iiitd.edu.in/raghava/vaxinpad/team.php) prediction portal. PIP-EL (http://www.thegleelab.org/PIP-EL/) prediction server for pro-inflammatory inducing peptides was used to predict the chance of Pp-ALF as a PIP.
Recombinant expression of P. pelagicus ALF
The mature peptide sequences of P. pelagicus ALF were amplified using primers designed with BamH1and EcoR1 restriction sites. The Amplicons after purification was inserted into pGEMTR–T easy cloning vector and digested with respective restriction enzymes (RE). pET 32a (+) was used as the expression vector. The RE-digested products were gel purified, and the PCR product was inserted into pET-32a (+) plasmid. This transformation produced the recombinant pET-32a-Pp ALF which was then sequenced with T7F (5′TGTAATACGACTCACTATAGGG3′) and T7R (5′CTAGTTATTGCTCAGCGGTG3′) primers. The pET-32a-Pp ALF plasmid was then transformed into the E. coli Rosetta-gamiTMB (DE3) p Lys S (Novagen, 231 Cricklewood Broadway, London, UK), the expression host. The amplification was confirmed by T7F and T7R primers. The parent vector without an insert fragment was selected as a negative control. After sequencing to ensure in-frame insertion, positive transformants and the negative control were incubated overnight in 10 ml Luria Bertani (LB) medium [containing ampicillin (50 mg/ml), kanamycin (15 mg/ml), chloramphenicol (12.5 mg/ml), and tetracycline (34 mg/ml)] at 37 °C at 250 rpm in an Incubator Shaker (JeioTech, Korea). This was added as inoculum into 250 ml LB medium with the above mentioned antibiotics and incubated at 37 °C at 250 rpm. When the culture reached OD600 of 0.5-0.7, IPTG (isopropyl-β-D-thiogalactosidase) was added to a final concentration of 1 mM and incubated. Two ml aliquots of the cultures were taken every hour after induction, centrifuged, and the cell pellet was kept at −20 °C for the detection of the recombinant peptide by 15% SDS-PAGE examination.
Cytotoxic effect of the peptide Pp-ALF on the expression host E. coli in the production medium (live/dead assay)
SYTO 9–propidium iodide (PI) staining
Since the turbidity of the production medium decreased post-induction and the peptide production could not be detected by SDS- PAGE, the viability of the host cells was checked by SYTO9 - PI staining. The assay was used to analyze the live (membrane intact)/dead (membrane compromised) condition of the expression host E. coli Rosetta-gami, due to the production of the peptide Pp-ALF. E. coli transformed with the pET-32a (+) vector without insert was kept as a negative control, which expresses thioredoxin (Trx). The dye suspension (SYTO 9: PI; 1:1) was added to the cell pellets collected at 0, 1, 2, 4, and 6th-hour post-induction, incubated for 15 min in dark at room temperature and observed under Epifluorescence Microscope ( Carl Zeiss Axio observer) with Ex/Em 480/500 nm for SYTO 9 and 490/635 nm for PI.
Triple staining (CTC-DAPI-FITC)
In order to observe the cells, both live/dead and membrane permeabilization caused by the peptide, if any in the expression host, triple-staining was done . Fluorochromes used were (i) the double-stranded DNA-binding dye DAPI (stain all bacterial cells both live/dead), (ii) the vital dye CTC (stain only viable cells), and (iii) FITC that traverse only permeabilized cytoplasmic membrane. Both CTC and DAPI were made up as stock solutions in water at a concentration of 6 mg/ml (20 mM) and 10 mg/ml (28 mM), respectively. After induction with IPTG, the host cells (Rosetta-gami pET-32a-Pp-ALF) were sampled at 0, 2, and 6 and incubated with 900 μl of CTC for 90 min at 37 °C. An aliquot (10 μl) of the reaction mix was transferred to a poly L-lysine-coated glass slide. The slides were washed with sodium phosphate buffer, and 1ml of DAPI solution (10 μg/ml in PBS) was added and after 30 min at 30 °C. The DAPI solution was removed and the slides were rinsed again with sodium phosphate buffer. To the cells, FITC was added (6 mg/ml in sodium phosphate buffer) and incubated at 30 °C for 30 min and washed as mentioned above. The slides were then examined under a Fluorescence microscope (Carl Zeiss, Germany).
Molecular characteristics of Pp-ALF
A 369bp nucleotide sequence coding the complete cDNA sequence of ALF encoding 123 amino acids (Fig. 1) obtained from mRNA of P. pelagicus hemocyte by reverse transcription. Hereafter, the ALF is referred to as Pp-ALF (GenBank ID: OP009359). A similarity search using BLASTn and BLASTp algorithms confirmed the cDNA sequence coming under the ALF family of antimicrobial peptides. Upon BLASTp homology search Pp-ALF showed 95.89 % similarity with Portunus trituberculatus ALF (GenBank ID: ADU25043.1) followed by Scylla paramamosain ALF (GenBank ID: AFI43796.1) with 89.04% similarity. The putative signal peptide was identified at the N-terminal sequence, defined by SignalP software, with the cleavage site between amino acid positions 26 and 27 (CEA-QY). The signal peptide is followed by a highly cationic 97 amino acid mature peptide sequence comprising 22 amino acid long lipopolysaccharide (LPS) binding domain.
Pp-ALF was found to have a predicted molecular weight of 14.053 kDa, a net charge of +10.75 and a theoretical isoelectric point (pI) of 9.8. The mature region of the sequence has aforesaid parameters as 11.29 kDa, +9.75, and 10.17, respectively. The cationic nature of the Pp-ALF was mainly contributed by Lys (7%), Arg (9%), and His (2%) against negatively charged amino acids like Glu (3%) and Asp (6%). The predicted half-life of Pp-ALF in mammalian reticulocytes is about 30h, in vitro and >10h in E. coli, in vivo. The grand average hydropathy value (GRAVY) of the peptide was estimated to be −0.29 and the Wimley white whole residue hydrophobicity was 12.87. Kyte & Doolittle plot revealed the hydrophobicity; the residues from 5 to 21 (-VVAGLCLALVVMCLYLP-) in the signal region exhibited the highest hydrophobicity (Fig. 2) which shows the importance of the signal peptide domain for the protein translocation. Residues 28 to 40 (-YDALVASILGKLT-) showed the highest hydrophobicity in the mature domain. The amino acid distribution resulting in amphipathicity is illustrated by the helical wheel diagram (Fig. 3). Polar residues including glycine made up 50% of the predicted total, whereas non-polar residues made up the remaining 50%. The peptide’s hydrophobicity was 0.378 H, with a hydrophobic moment of 0.060μH and the hydrophobic phase is supplied by -FWLP- residues.
Multiple sequence alignment and phylogenetic analysis
The multiple sequence alignments of Pp-ALF with other crustacean ALFs retrieved from GenBank revealed the conserved motifs and residues. The two cysteine residues (Cys55-Cys76) involved in the internal disulfide bond formation were totally conserved in Pp-ALF (Fig. 4). Pp-ALF possessed the highest similarity with P. trituberculatus (ADU25043.1) (95.89%) in terms of amino acid composition. Pp-ALF has Ala at the 33rd position, which is substituted by Thr in P. trituberculatus, similarly, Thr is substituted with Ile at the 54th position. A consensus pattern of WCPGWA (T) was also observed in all ALFs. The evolutionary relationships with other crustacean lineages were found by phylogenetic analysis. Bootstrapped method using deduced amino acid sequences was used to elucidate the ancestral relationships (Fig. 5). The tree forms three groups, group 1 includes ALFs from shrimps, group 2 comprises ALF from cray fishes, and group 3 consists of crab ALFs including the Pp-ALF from P. pelagicus.
Pp-ALF had a predicted mRNA structure with minimum free energy (MFE) of −123.30 kcal/mol. This indicates the structural stability of the stem-loop structure of mRNA (Fig. 6). The nucleotides were mostly paired and formed intramolecular base pairing of RNA. The secondary structure of Pp-ALF showed an α helical structure in N-terminal signal peptide region followed by the other two helices towards C-terminal of the mature region. Between these helices consists of segments of β strands and randomly coiled structure. The amphipathic LPS binding domain was formed by β strands and coiled segments (Fig. 7). The spatial orientation of the Pp-ALF was constructed using the solution structure of an ALF (c2jobA) as template. The template shared 100% confidence with Pp-ALF. The 3D structure (Fig. 8) consists of three α helices packed against four β sheets. The β sheets, 1 (His 53-Arg 64) and 2 (Lys 67-Trp 75) are linked by a conserved disulphide bond (Cys55-Cys 76) forming the highly cationic (+7.25, pI 10.54) and amphipathic β hairpin loop structure, the LPS binding domain. The highly cationic LPS domain permits the peptide to effectively interact with negatively charged bacterial membranes. Structure validation of Pp-ALF by Ramachandran plot (Fig. 9) showed 85.2% residues in the most allowed region, 13.6% in the additionally allowed area, and 1.1% amino acid residues in the generously allowed region. No residues were found in the disallowed region. Based on the analysis, the R-factor is no greater than 20% and a good-quality model is expected to have over 90% residues in the allowed regions.
CAMP server predicted Pp-ALF as an antimicrobial peptide using support vector machine (SVM) classifier. iAMPpred identified Pp-ALF as antibacterial, antiviral, and antifungal with 0.96, 0.96, and 0.98 SVM score, respectively. SVM score near to 1 is identified as a peptide with high therapeutic potential. Even though Pp-ALF was predicted to exhibit antimicrobial potential, the region with high aggregation propensity towards microbial membrane was predicted by AGGRESCAN. There are two hotspots predicted for Pp-ALF; the first hotspot (HS) area with a normalized value of 0.745 was found inside the signal peptide domain (GVVAGLCLALVVMCLYL), and the second HS was inside the mature region (DALVASILGKLTGL) with a normalized value of 0.347.
The LPS domain of Pp-ALF was predicted with anti-angiogenic activity by “AntiAngioPred” with an SVM score of 0.82 indicating its anticancer property. Also, the average antigenic propensity of Pp-ALF was found as 1.0366 by the “Immunomedicine group.” The server predicted 5 antigenic sequence regions in Pp-ALF, i.e., 4 to 38 residues (GVVAGLCLALVVMCLYLPQPCEAQYDALVASILGK), 49 to 57 (DFMGHTCYF), 64 to 70 (RRFKLYH), 72 to78 (GKFWCPG), and 112 to119 (ITQQDASL). So, Pp-ALF could be used as an immunogen with prediction values of 0.316 (AntiTbpred) and 0.574 (AIPpred) as anti-tubercular and anti-inflammatory peptide. Biofilms provide survival sites for opportunistic pathogens. The LPS domain of Pp-ALF was predicted as an antibiofilm agent (SVM score −0.83). The ability to distinguish between an antigenic cell and host cell is an important property of an ideal AMP. HemoPred predicted the LPS domain of Pp-AF as non-hemolytic. This property leads to the safe application of the peptide in the biological system.
Peptides are divided into two types based on their amino acid composition; cell-penetrating and non-cell-penetrating peptides. Pp-ALF was predicted to be CPP (cell-penetrating peptide) with a positive SVM score of 0.01. CPPs are made up of basic amino acids like arginine and lysine, and they can cross membranes to obtain access to the cell’s core. Pp-ALF consists of a total of 17 arginine and lysine residues in the mature region of the peptide. This was confirmed by “DNA binder”, which gave the Pp-ALF a positive score of 1.25, indicating it as a DNA-binding protein. Pp-ALF possesses immunomodulatory activity as predicted by “VaxinPAD” with an SVM value of 0.97. So, Pp-ALF has the potential to stimulate the innate immune system as an adjuvant. In immunotherapy, pro-inflammatory peptides (PIP) have been employed as anticancer agents, antibacterial agents, and vaccines. Pp-ALF has been predicted as pro-inflammatory peptide (PIP) with a probability score of 0.7633. The projected value of the codon adaptation index, which represents the likely success of heterologous gene expression was, 0.57 in Escherichia coli, 0.58 in Saccharomyces cerevisiae, and 0.6 in Pichia pastoris. The peptide rank score was calculated to be 0.99, indicating the high bioactive potential of Pp-ALF.
Recombinant expression of Pp-ALF and cytotoxicity to the expression host
Live/dead assay by SYTO 9-PI staining of cells
Turbidity of the production medium with the expression host E. coli (pET-32a- Pp-ALF) was found decreasing post-induction with IPTG. The cell lysate of the expression host E. coli when analyzed by SDS-PAGE did not display the presence of the recombinant peptide Pp-ALF (29.08kDa with Trx tag) (Fig. 10). However, thioredoxin (Trx 17.72 kDa) could be detected in control (expression host E. coli with pET-32a) (Fig. 11).
SYTO 9 - propidium iodide (PI) staining of the host cells revealed the death of the cells during the production of Pp-ALF. The recombinantly expressed Pp-ALF causes the death of the expression host, and this was observed on staining. The cells were green fluorescent at the 0th hour since they were metabolically active. From the 1st hour post-induction itself, the cytotoxic effect on host cells was clearly visible and from the 2nd hour onwards complete cell death was confirmed by red fluorescence due to the internalization of propidium iodide stain (Figs. 12 and 13). Apart from this, the bacteria form a mucilaginous sheath in response to the peptide production. In the case of control, cells were active throughout the production period (5 h post-induction).
Live/dead/membrane permeabilizationby CTC-DAPI-FITC staining
Cell death as well as permeabilization was not noticed initially (at the 0th h) at the time of IPTG induction as evidenced and confirmed by CTC-DAPI–FITC staining. Reduction in viable cells and membrane permeabilization was observed at the 2nd h. Viable cells were not observed at the 6th h, and only membrane-permeabilized/dead cells were detected. This concludes that the peptide Pp-ALF causes death to the host cells at the initial stages of production itself. The peptide also causes a high degree of cellular clumping which was also observed in cells at the 6th h (Fig. 14).
Due to the emergence of antibiotic-resistant microbes, the significance of alternate compounds like AMPs to combat infections has been increased . In the present study, an anti-lipopolysaccharide factor Pp-ALF was cloned from P. pelagicus. An essential physicochemical property of AMPs is their hydrophobicity [23,24,25,26]. Pp-ALF possesses a consensus pattern of “WCPGWT”, besides the highly conserved pair of cysteine residues and the LPS-binding domain [15, 27]. The newly discovered Pp-ALF has three helices packed against four β sheets, which is similar to the 3D structure of LALF (Limulus ALF), ALFPm3, (Penaeus mondon) SpALF4 (Scylla paramamosain), and SpALF7 [28,29,30,31]. The disulfide bond is crucial for the stability of the 3D structure of ALF . On phylogenetic analysis, the ALF sequences were found clustering into species-specific groups. The ALFs from crabs, shrimps, and crayfishes were all grouped independently. A further finding from the data is that Pp-ALF has a stronger association with crab ALFs than shrimp and crayfish ALFs.
The LPS-binding domain of Pp-ALF possesses 8 hydrophobic amino acids including one tryptophan. It has been demonstrated that the ALFs with high pI value, bind with LPS inhibiting the proliferation of Gram-negative bacteria [32,33,34]. Inside the amphipathic loop of LPS in Pp-ALF, nine positively charged amino acid residues are present with a high pI of 10.54. Rosa et al.  reported that ALF with higher isoelectric point possesses high antibacterial activity. In S. paramamosain, SpALF1 and SpALF2 with high pI showed antimicrobial activity against both Gram-negative and Gram-positive bacteria .
The biological functions of ALFs mainly depend on the LPS domain that interacts with the negatively charged LPS . Positively charged amino acids (arginine and lysine) in the LPS domain contribute to the antibacterial activity of the organisms and the activities were found to diminish, if the arginine and lysine residues were substituted with neutral amino acids [5, 37, 38]. Substitution of lysine in a parotid secretory protein, GL13NH2 changed its functional property from bacterial agglutinating peptide to bactericidal peptide . Due to the high prevalence of lysine and/or arginine in most AMPs, their net charges range from +2 to +11 [40, 41]. Pp-ALF had a net charge of +10.17 for the mature peptide indicating the high cationicity of the peptide. It is generally accepted that the initial interaction of the AMP with the negatively charged membrane surface of the bacterium is predominantly caused by cationicity [42, 43]. LPS domain of Pp-ALF embedded with five arginine and three lysine residues contributing 23 and 14% of the total amino acids, respectively, in the LPS region indicating its potential role as an antimicrobial agent. FcALF2 (Fenneropenaeus chinensis ALF) and PmALF3 (Penaeus monodon ALF) with five and six positively charged amino acids in the LPS domain have shown strong antibacterial activity [35, 44]. Pp-ALF shares important characteristics of LALFs (Limulus ALF), with the hydrophobic N-terminal sequences and the concentration of positive charges in the disulphide loop that are crucial for their antimicrobial effect [45, 46].
Pp-ALF was found to possess 41% hydrophobicity as per APD analysis. Hydrophobicity controls the degree to which a peptide can partition into the lipid bilayers and therefore play an important role in its activity [47,48,49]. Hydrophobicity is necessary for membrane permeabilization and higher hydrophobicity results in higher antibacterial action [23, 50]. The hydrophobicity of sSpALF7 from Scylla paramamosain was higher (51.4%) than that of rSpALF7 (41.5%), which might be one of the reasons why sSpALF7 showed a broader antibacterial spectrum. Analysis of the Pp-ALF sequence using the Kyte-Doolittle plot exhibited a significant presence of hydrophobic amino acids concentrated in the first 17 residues (-VVAGLCLALVVMCLYLP-) of the mature peptide. This demonstrates that these amino acids are components of the alpha helix and may have the ability to traverse a lipid bilayer of the microbial membrane . Pp-ALF was predicted to possess antibacterial, antifungal, and antiviral activities, and it was found to have high therapeutic potential according to the prediction score. Six recombinant PtALF proteins could prevent the growth of specific Gram-positive, Gram-negative bacteria, or fungi in the swimming crab P. trituberculatus [34, 52].
ALF from P. monodon showed inhibition against herpes simplex virus type 1, human adenovirus respiratory strain, and WSSV replication [16, 53]. LBD peptides, i.e., FcALF1, FcALF2, FcALF5, and ALFFc from Fenneropenaeus chinensis could inhibit WSSV replication . Crab-ALF2A and crab-ALF6A, isolated from P. trituberculatus, showed minimal effective concentrations (MECs) of 2.11 μg/mL and 1.95 μg/mL against the yeast Candida albicans, respectively, demonstrating promising antiviral and antifungal activity against yeasts and viruses . ALFPm3 from black tiger shrimp P. monodon  showed antifungal and antibacterial activities. Bacterial cell membranes have been destroyed by mFcALF2 (modified ALF from Fenneropenaeus chinensis) resulting in the leakage of cytoplasm . MjALF-D from Marsupenaeus japonicas also exhibited antibacterial activity . Pp-ALF is predicted as a cell-penetrating peptide and therefore might cause pore formation resulting in the leakage of cell contents.
Synthetic SALF (shrimp ALF) from black tiger shrimp displayed anticancer properties in HeLa cells when tested in mice. SALF disrupted the tumor cell membrane triggering apoptosis . Pp-ALF is also predicted to have antiangiogenic properties by the Anticp and antiangiopred server with a significant prediction score.
The majority of the studies have concentrated on the production of AMPs in bacteria due to the availability of well-defined plasmid vectors and the practical significance of large-scale peptide production through fermentation. However, since some of these peptides are toxic to the expression host, direct expression of AMPs is typically challenging . As a result, these peptides are produced as fusion proteins in bacterial strains lacking proteases, including E. coli BL21. The fusion approach enhances solubility and prevents toxicity and cell disintegration in the expression host. The recombinant expression has greater benefits than high-cost chemical synthesis and low-yield natural extraction. Due to the potent antibacterial properties displayed by Pp-ALF in silico, recombinant production was attempted using the E. coli expression system. However, the peptide was found to be toxic to the host cells since post induction with IPTG, the cells started dyeing which might be due to the production of the peptide Pp-ALF. rMnALF4 (recombinant ALF from Macrobrachium nipponense) also showed toxicity in E. coli expression system; so it was produced in P. pastoris with a high yield. Additionally, Salmonella typhi, Shigella sp., Staphylococcus aureus, Pseudmonas fluorescens, and Salmonella gullinarum, all of which are extremely pathogenic for humans, were sensitive to rMnALF4 . Pp-ALF production was toxic to the host, which was proved by live dead SYTO 9-PI staining and triple staining. Death of the expression host was observed from the first hour onwards after IPTG induction.
The eukaryotic yeast expression host, P. pastoris is widely used in the heterologous expression of antimicrobial peptides, because of its potent secretion and glycosylation property. Anti-lipopolysaccharide factors from P. mondon , Macrobrachium rosenbergii , Macrobrachium nipponense , and Litopenaeus vannamei  were successfully expressed in the yeast expression system, P. pastoris. ALF from M. nipponense could not be expressed in E. coli , and in the present study also, Pp-ALF could not be expressed in E. coli. The reason for this differential antibacterial effect displayed by E. coli could not be explained.
Recombinant proteins can take on specific spatial structures and post-translational modifications when they are expressed by the eukaryotic expression system. These changes would better replicate the recombinant proteins’ natural state in living organisms, and the structure and modification are crucial for protein function . For the synthesis of AMPs, especially those with significant bacterial inhibitory action, the prokaryotic expression system, such as the E. coli system, is not typically used [59, 61]. Although the precise processes by which AMPs exert their microbicidal action are not yet fully understood, it is generally agreed that AMPs primarily target the cytoplasmic membrane through penetration and cell lysis activities . Conditional toxicity is the main issue in this situation; specifically, the AMP should not be toxic to the expression host while it is forming, but should be active when employed to combat the infecting bacterium . Recombinantly generated peptides lack this property and are also vulnerable to enzymatic digestion and lack post-translational modification. Recombinant proteins can be successfully synthesized as secretory proteins in the yeast P. pastoris expression system when a signal peptide is linked to the foreign protein at its N-terminus . Since Pp-ALF was toxic to E. coli cells, an alternate expression system like P. pastoris would be a viable option. The success of heterologous expression predicted by the codon adaptation index showed that P. pastoris system has the highest chance for expression of Pp-ALF. Synthetic biology is also another option for the synthesis of these molecules . The use of bioactive peptides in the development of novel medical therapies would be highly promising. This work provides concise information with regard to an AMP, anti-lipopolysaccharide factor characterized from P. pelagicus with potent antimicrobial activity predicted in silico and displayed by the death of the host E. coli DH5 alpha cells on heterologous expression.
Functional prediction of the anti-lipopolysaccharide factor from P. pelagicus, Pp-ALF revealed that it could be a potent molecule with respect to antimicrobial, anti-inflammatory, and anticancer activity. The prokaryotic expression system E. coli was not found suitable for the recombinant expression of Pp-ALF due to its cytotoxic effect on the host cells. Eukaryotic expression systems would be a preferred option for the recombinant production of Pp-ALF.
Availability of data and materials
The data generated during and/or analyzed during the current study are not publicly available (except GenBank accession) but are available from the corresponding author on reasonable request.
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ErdemBüyükkiraz M, Kesmen Z (2022) Antimicrobial peptides (AMPs): a promising class of antimicrobial compounds. J Appl Microbiol 132(3):1573–1596. https://doi.org/10.1111/jam.15314
Salyers AA, Amabile-Cuevas CF (1997) Why are antibiotic resistance genes so resistant to elimination? Antimicrob Agents Chemother 41(11):2321–2325. https://doi.org/10.1128/AAC.41.11.2321
Saucedo-Vázquez JP, Gushque F, Vispo NS, Rodriguez J, Gudiño-Gomezjurado ME, Albericio F, Tellkamp MP, Alexis F (2022) Marine arthropods as a source of antimicrobial peptides. Mar Drugs 20(8):501. https://doi.org/10.3390/md20080501
Zhang H, Zheng J, Cheng W, Mao Y, Yu X (2022) Antibacterial activity of an anti-lipopolysaccharide factor (MjALF-D) identified from kuruma prawn (Marsupenaeus japonicus). Fish Shellfish Immunol 127:295–305. https://doi.org/10.1016/j.fsi.2022.06.036
Li S, Guo S, Li F, Xiang J (2014) Characterization and function analysis of an anti-lipopolysaccharide factor (ALF) from the Chinese shrimp Fenneropenaeuschinensis. Dev Comp Immunol l46(2):349–355. https://doi.org/10.1016/j.dci.2014.05.013
Morita T, Ohtsubo S, Nakamura T, Tanaka S, Iwanaga S, Ohashi K, Niwa M (1985) Isolation and biological activities of limulus anticoagulant (AntiLPS factor) which interacts with lipopolysaccharide (LPS). J Biochem 97(6):1611–1620
Muta T, Miyata T, Tokunaga F, Nakamura T, Iwanaga S (1987) Primary structure of anti-lipopolysaccharide factor from American horseshoe crab, Limulus polyphemus. J Biochem 101(6):1321–1330. https://doi.org/10.1093/oxfordjournals.jbchem.a121999
Yin C, Shen X, Wang Y, Hu J, Bao Z, Wang M (2023) Comparative study of five anti-lipopolysaccharide factor genes in Litopenaeus vannamei. Dev Comp Immunol 139:104557. https://doi.org/10.1016/j.dci.2022.104557
Sun M, Li S, Lv X, Xiang J, Lu Y, Li F (2021) A lymphoid organ specific anti-lipopolysaccharide factor from Litopenaeus vannamei exhibits strong antimicrobial activities. Mar Drugs 19(5):250. https://doi.org/10.3390/md19050250
Anju MV, Archana K, Nair A, Philip R (2020) An anti-lipopolysaccharide factor Md-ALF from the Indian flower tail shrimp, Metapenaeus dobsoni: molecular and phylogenetic characterization. Gene Rep 21:100867. https://doi.org/10.1016/j.genrep.2020.100867
Zhu JJ, Ye ZZ, Li CS, Kausar S, Abbas MN, Xiang GH, Qian XY, Dai LS (2019) Identification and molecular characterization of a novel anti-lipopolysaccharide factor (ALF) from red swamp crayfish, Procambarus clarkii. Int J Biol Macromol 132:43–50. https://doi.org/10.1016/j.ijbiomac.2019.03.167
Beale KM, Towle DW, Jayasundara N, Smith CM, Shields JD, Small HJ, Greenwood SJ (2008) Anti-lipopolysaccharide factors in the American lobster Homarus americanus: molecular characterization and transcriptional response to Vibrio fluvialis challenge. Comp Biochem Physiol Part D Genomics Proteomics 3(4):263–269. https://doi.org/10.1016/j.cbd.2008.07.001
Nam BH, Park EH, ShinE H, Kim YO, Kim DG, Kong HJ, Park JY, Seo JK (2019) Development of novel antimicrobial peptides derived from anti-lipopolysaccharide factor of the swimming crab, Portunus trituberculatus. Fish Shellfish Immunol 84:664–672. https://doi.org/10.1016/j.fsi.2018.10.031
Sruthy KS, Philip R (2021) Anti-lipopolysaccharide factor from crucifix crab Charybdis feriatus, Cf-ALF2: molecular cloning and functional characterization of the recombinant peptide. Probiotics Antimicrob Proteins 13(3):885–898. https://doi.org/10.1007/s12602-020-09716-w
Liu Y, Cui Z, Li X, Song C, Shi G, Wang C (2013) Molecular cloning, genomic structure and antimicrobial activity of PtALF7, a unique isoform of anti-lipopolysaccharide factor from the swimming crab Portunus trituberculatus. Fish Shellfish Immunol 34(2):652–659. https://doi.org/10.1016/j.fsi.2012.12.002
Tharntada S, Ponprateep S, Somboonwiwat K, Liu H, Söderhäll I, Söderhäll K, Tassanakajon A (2009) Role of anti-lipopolysaccharide factor from the black tiger shrimp, Penaeus monodon, in protection from white spot syndrome virus infection. J Gen Virol 90(6):1491–1498. https://doi.org/10.1099/vir.0.009621-0
Tang T, Liu J, Li S, Li H, Liu F (2020) Recombinant expression of an oriental river prawn anti-lipopolysaccharide factor gene in Pichia pastoris and its characteristic analysis. Fish Shellfish Immunol 98:414–419. https://doi.org/10.1016/j.fsi.2020.01.030
Zainal KA (2013) Natural food and feeding of the commercial blue swimmer crab, Portunus pelagicus (Linnaeus, 1758) along the coastal waters of the Kingdom of Bahrain. J Assoc Arab Univ Basic Appl Sci 13(1):1–7. https://doi.org/10.1016/j.jaubas.2012.09.002
Kunsook C, Gajaseni N, Paphavasit N (2014) The feeding ecology of the blue swimming crab, Portunus pelagicus (Linnaeus, 1758), at Kung Krabaen Bay, Chanthaburi Province, Thailand. Trop Life Sci Res 25(1):13 PMID: 25210585; PMCID: PMC4156471
Haputhantri SSK, Bandaranayake KHK, Rathnasuriya MIG, Nirbadha KGS, Weerasekera SJWWMMP, Athukoorala AASH, Jayathilaka RAM, Perera HACC, Creech S (2022) Reproductive biology and feeding ecology of the blue swimming crab (Portunuspelagicus) in Northern Coastal Waters, Sri Lanka. Trop Life Sci Res 33(2):155. https://doi.org/10.21315/tlsr2022.33.2.8
Mangoni ML, Papo N, Barra D, Simmaco M, Bozzi A, Di Giulio A, Rinaldi AC (2004) Effects of the antimicrobial peptide temporin L on cell morphology, membrane permeability and viability of Escherichia coli. Biochem J 380(3):859–865. https://doi.org/10.1042/BJ20031975
Jiang HS, Lv LX, Wang JX (2022) Anti-lipopolysaccharide factor D from kuruma shrimp exhibits antiviral activity. Mar Life Sci Technol 4:52–61. https://doi.org/10.1007/s42995-021-00113-y
Yount NY, Yeaman MR (2005) Immunocontinuum: perspectives in antimicrobial peptide mechanisms of action and resistance. Protein Pept Lett 12(1):49–67. https://doi.org/10.2174/0929866053405959
Pasupuleti M, Chalupka A, Mörgelin M, Schmidtchen A, Malmsten M (2009a) Tryptophan end-tagging of antimicrobial peptides for increased potency against Pseudomonas aeruginosa. Biochim Biophys Acta Gen Subj 1790(8):800–808. https://doi.org/10.1016/j.bbagen.2009.03.029
Pasupuleti M, Davoudi M, Malmsten M, Schmidtchen A (2009b) Antimicrobial activity of a C-terminal peptide from human extracellular superoxide dismutase. BMC Res 2(1):1–6. https://doi.org/10.1186/1756-0500-2-136
Schmidtchen A, Pasupuleti M, Mörgelin M, Davoudi M, Alenfall J, Chalupka A, Malmsten M (2009) Boosting antimicrobial peptides by hydrophobic oligopeptide end tags. J Biol Chem 284(26):17584–17594. https://doi.org/10.1074/jbc.M109.011650
Wu X, Huang Y, Yu Z, Mu C, Song W, Li R, Liu L, Ye Y, Shi C, Wang C (2019) An MBT domain containing anti-lipopolysaccharide factor (PtALF8) from Portunus trituberculatus is involved in immune response to bacterial challenge. Fish Shellfish Immunol 84:252–258. https://doi.org/10.1016/j.fsi.2018.10.016
Hoess A, Watson S, Siber GR, Liddington R (1993) Crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, Limulus anti-LPS factor, at 1.5 A resolution. EMBO J 12(9):3351–3356. https://doi.org/10.1002/j.1460-2075.1993.tb06008.x
Yang Y, Boze H, Chemardin P, Padilla A, Moulin G, Tassanakajon A, Pugnière M, Roquet F, Destoumieux-Garzón D, Gueguen Y, Bachère E, Aumelas A (2009) NMR structure of Ralf-Pm3, an anti-lipopolysaccharide factor from shrimp: model of the possible lipid -binding site. Biopolymers Orig Res Biomol 91(3):207–220. https://doi.org/10.1002/bip.21119
Zhu L, Lan JF, Huang YQ, Zhang C, Zhou JF, Fang WH, Yao XJ, Wang H, Li XC (2014) SpALF4: a newly identified anti-lipopolysaccharide factor from the mud crab Scylla paramamosain with broad spectrum antimicrobial activity. Fish Shellfish Immunol 36(1):172. https://doi.org/10.1016/j.fsi.2013.10.023
Long S, Chen F, Wang KJ (2021) Characterization of a new homologous anti-lipopolysaccharide factor SpALF7 in mud crab Scylla paramamosain. Aquac 534:736333. https://doi.org/10.1016/j.aquaculture.2020.736333
Methatham T, Boonchuen P, Jaree P, Tassanakajon A, Somboonwiwat K (2017) Antiviral action of the antimicrobial peptide ALFPm3 from Penaeus monodon against white spot syndrome virus. Dev Comp Immunol 69:23–32. https://doi.org/10.1016/j.dci.2016.11.023
Liu Y, Cui Z, Luan W, Song C, Nie Q, Wang S, Li Q (2011) Three isoforms of anti-lipopolysaccharide factor identified from eyestalk cDNA library of swimming crab Portunus trituberculatus. Fish Shellfish Immunol 30(2):583–591. https://doi.org/10.1016/j.fsi.2010.12.005
Liu Y, Cui Z, Li X, Song C, Li Q, Wang S (2012) A new anti-lipopolysaccharide factor isoform (PtALF4) from the swimming crab Portunus trituberculatus exhibited structural and functional diversity of ALFs. Fish Shellfish Immunol 32(5):724–731. https://doi.org/10.1016/j.fsi.2012.01.021
Rosa RD, Vergnes A, de Lorgeri J, Goncalves P, Perazzolo LM, Saune L, Romestand B, Fievet J, Gueguen Y, Bachere E, Destoumieux-Garzon D (2013) Functional divergence in shrimp anti-lipopolysaccharide factors (ALFs): from recognition of cell wall components to antimicrobial activity. PLoS One 8(7):e67937. https://doi.org/10.1371/journal.pone.0067937
Sun W, Wan W, Zhu S, Wang S, Wang S, Wen X, Zheng H, Zhang Y, Li S (2015) Characterization of a novel anti-lipopolysaccharide factor isoform (SpALF5) in mud crab, Scylla paramamosain. Mol Immunol 64(2):262–275. https://doi.org/10.1016/j.molimm.2014.12.006
Guo S, Li S, Li F, Zhang X, Xiang J (2014) Modification of a synthetic LPS-binding domain of anti-lipopolysaccharide factor from shrimp reveals strong structure-activity relationship in their antimicrobial characteristics. Dev Comp Immunol 45(2):227–232. https://doi.org/10.1016/j.dci.2014.03.003
Lv X, Li S, Liu F, Li F, Xiang J (2017) Identification and function analysis of an anti-lipopolysaccharide factor from the ridgetail prawn Exopalaemon carinicauda. Dev Comp Immunol 70:128–134. https://doi.org/10.1016/j.dci.2017.01.010
Abdolhosseini M, Nandula SR, Song J, Hirt H, Gorr SU (2012) Lysine substitutions convert a bacterial-agglutinating peptide into a bactericidal peptide that retains anti-lipopolysaccharide activity and low hemolytic activity. Peptides 35(2):231–238. https://doi.org/10.1016/j.peptides.2012.03.017
Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55(1):27–55. https://doi.org/10.1124/pr.55.1.2
Niyonsaba F, Nagaoka I, Ogawa H (2006) Human defensins and cathelicidins in the skin: beyond direct antimicrobial properties. Crit Rev Immunol 26(6):545–576. https://doi.org/10.1615/CritRevImmunol.v26.i6.60
Yount NY, Yeaman MR (2006) Structural congruence among membrane-active host defense polypeptides of diverse phylogeny. Biochim Biophys Acta Biomembr 1758(9):1373–1386. https://doi.org/10.1016/j.bbamem.2006.03.027
Malanovic N, Lohner K (2016) Gram-positive bacterial cell envelopes: the impact on the activity of antimicrobial peptides. Biochim Biophys Acta Biomembr 1858(5):936–946. https://doi.org/10.1016/j.bbamem.2015.11.004
Li S, Guo S, Li F, Xiang J (2015) Functional diversity of anti-lipopolysaccharide factor isoforms in shrimp and their characters related to antiviral activity. Mar Drugs 13(5):2602–2616. https://doi.org/10.3390/md13052602
Imjongjirak C, Amparyup P, Tassanakajon A (2011) Molecular cloning, genomic organization and antibacterial activity of a second isoform of antilipopolysaccharide factor (ALF) from the mud crab, Scylla paramamosain. Fish Shellfish Immunol 30(1):58–66. https://doi.org/10.1016/j.fsi.2010.09.011
Sun C, Xu WT, Zhang HW, Dong LP, Zhang T, Zhao XF, Wang JX (2011) An anti-lipopolysaccharide factor from red swamp crayfish, Procambarus clarkii, exhibited antimicrobial activities in vitro and in vivo. Fish Shellfish Immunol 30(1):295–303. https://doi.org/10.1016/j.fsi.2010.10.022
Dennison SR, Harris F, Phoenix DA (2005) Are oblique orientated α-helices used by antimicrobial peptides for membrane invasion? Protein Pept Lett 12(1):27–29. https://doi.org/10.2174/0929866053406039
Ringstad L, Nordahl EA, Schmidtchen A, Malmsten M (2007) Composition effect on peptide interaction with lipids and bacteria: variants of C3a peptide CNY21. Biophys J 92(1):87–98. https://doi.org/10.1529/biophysj.106.088161
Ringstad L, Protopapa E, Lindholm-Sethson B, Schmidtchen A, Nelson A, Malmsten M (2008) An electrochemical study into the interaction between complement-derived peptides and DOPC mono-and bilayers. Langmuir 24(1):208–216. https://doi.org/10.1021/la702538
Jiang Z, Kullberg BJ, van der Lee H, Vasil AI, Hale JD, Mant CT, Hancock RE, Vasil ML, Netea MG, Hodges RS (2008) Effects of hydrophobicity on the antifungal activity of alpha-helical antimicrobial peptides. Chem Biol Drug Des 72:483–495. https://doi.org/10.1111/j.1747-0285.2008.00728.x
Yedery RD, Reddy KVR (2009) Identification, cloning, characterization and recombinant expression of an anti-lipopolysaccharide factor from the hemocytes of Indian mud crab, Scylla serrata. Fish Shellfish Immunol 27(2):275–284. https://doi.org/10.1016/j.fsi.2009.05.009
Liu Y, Cui Z, Li X, Song C, Shi G (2013) A newly identified anti-lipopolysaccharide factor from the swimming crab Portunus trituberculatus with broad spectrum antimicrobial activity. Fish Shellfish Immunol 34(2):463–470. https://doi.org/10.1016/j.fsi.2012.11.050
Carriel-Gomes MC, Kratz JM, Barracco MA, Bachére E, Barardi CRM, Simões CMO (2007) In vitro antiviral activity of antimicrobial peptides against herpes simplex virus 1, adenovirus, and rotavirus. Mem Inst Oswaldo Cruz 102:469–472. https://doi.org/10.1590/S0074-02762007005000028
Somboonwiwat K, Bachère E, Rimphanitchayakit V, Tassanakajon A (2008) Localization of anti-lipopolysaccharide factor (ALFPm3) in tissues of the black tiger shrimp, Penaeus monodon, and characterization of its binding properties. Dev Comp Immunol 32(10):1170–1176. https://doi.org/10.1016/j.dci.2008.03.008
Yang H, Li S, Li F, Yu K, Yang F, Xiang J (2016) Recombinant expression of a modified shrimp anti-lipopolysaccharide factor gene in Pichia pastoris GS115 and its characteristic analysis. Mar Drugs 14(8):152. https://doi.org/10.3390/md14080152
Lin MC, Lin SB, Chen JC, Hui CF, Chen JY (2010) Shrimp anti-lipopolysaccharide factor peptide enhances the antitumor activity of cisplatin in vitro and inhibits HeLa cells growth in nude mice. Peptides 31(6):1019–1025. https://doi.org/10.1016/j.peptides.2010.02.023
Kim JM, Jang SA, Yu BJ, Sung BH, Cho JH, Kim SC (2008) High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage. Appl Microbiol Biotechnol 78:123–130. https://doi.org/10.1007/s00253-007-1273-5
Somboonwiwat K, Marcos M, Tassanakajon A, Klinbunga S, Aumelas A, Romestand B, Gueguen Y, Boze H, Moulin G, Bachère E (2005) Recombinant expression and anti-microbial activity of anti-lipopolysaccharide factor (ALF) from the black tiger shrimp Penaeus monodon. Dev Comp Immunol 29(10):841–851. https://doi.org/10.1016/j.dci.2005.02.004
Liu CC, Chung CP, Lin CY, Sung HH (2014) Function of an anti-lipopolysaccharide factor (ALF) isoform isolated from the hemocytes of the giant freshwater prawn Macrobrachium rosenbergii in protecting against bacterial infection. J Invertebr Pathol 116:1–7. https://doi.org/10.1016/j.jip.2013.12.001
Wu J, Lei K, Wu Z, Zhang Y, Gao W, Zhang W, Mai K (2022) Effects of recombinant anti-lipopolysaccharide factor expressed by Pichia pastoris on the growth performance, immune response and disease resistance of Litopenaeus vannamei. Fish Shellfish Immunol 129:231–242. https://doi.org/10.1016/j.fsi.2022.08.074
Ponprateep S, Somboonwiwat K, Tassanakajon A (2009) Recombinant anti-lipopolysaccharide factor isoform 3 and the prevention of vibriosis in the black tiger shrimp, Penaeus monodon. Aquac 289(3-4):219–224. https://doi.org/10.1016/j.aquaculture.2009.01.026
Chung PY, Khanum R (2017) Antimicrobial peptides as potential anti-biofilm agents against multidrug-resistant bacteria. J Microbiol Immunol Infect 50(4):405–410. https://doi.org/10.1016/j.jmii.2016.12.005
Pasupuleti M, Schmidtchen A, Malmsten M (2012) Antimicrobial peptides: key components of the innate immune system. Crit Rev Biotechnol 32(2):143–171. https://doi.org/10.3109/07388551.2011.594423
Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24(1):45–66. https://doi.org/10.1111/j.1574-6976.2000.tb00532.x
Jiménez JJ, Borrero J, Gútiez L, Arbulu S, Herranz C, Cintas LM, Hernández PE (2014) Use of synthetic genes for cloning, production and functional expression of the bacteriocinsenterocin A and bacteriocin E 50-52 by Pichia pastoris and Kluyveromyces lactis. Mol Biotechnol 56(6):571–583. https://doi.org/10.1007/s12033-014-9731-7
The authors are grateful to the Director, Centre for Marine Living Resources and Ecology (CMLRE), and Ministry of Earth Sciences (MoES), Govt. of India, for the research grant (MoES/10-MLR/01/2012) and scientific support for the work. The authors also thank the Director, National Centre for Aquatic and Animal Health (NCAAH), for the scientific/technical support of the work. The first author gratefully acknowledges the DST (Department of Science and Technology), Govt. of India, for the award of INSPIRE fellowship and the corresponding author to the University grants Commission, Government of India, for the BSR Faculty Grant (F.18 - 1/2011 (BSR) 2019).
This research was funded by the Ministry of Earth Sciences (MoES), Govt. of India, (MoES/10-MLR/01/2012)
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Anju, M.V., Archana, K., Anooja, V.V. et al. A novel anti-lipopolysaccharide factor from blue swimmer crab Portunus pelagicus and its cytotoxic effect on the prokaryotic expression host, E. coli on heterologous expression. J Genet Eng Biotechnol 21, 22 (2023). https://doi.org/10.1186/s43141-023-00478-w
- Anti-lipopolysaccharide factor
- Portunus pelagicus
- Crustacean immunity
- Antimicrobial peptides