Expression and Purification of the Main Component Contained in Camel Milk and Its Antimicrobial Activities Against Bacterial Plant Pathogens

Abstract

Lactoferrin is the most dominant protein in milk after casein. This protein plays a crucial role in many biological processes including the regulation of iron metabolism, induction and modulation of the immune system, the primary defense against microorganisms, inhibiting lipid peroxidation and presenting antimicrobial activity against various pathogens such as parasites, fungi, bacteria, and viruses. The major antimicrobial effect of lactoferrin is related to its N-terminal tail where different peptides for instance lactoferricin and lactoferrampin which are important for their antimicrobial abilities are present. The growth rate of bacterial cells in camel milk is lower than that of the cow milk due to having more antimicrobial compounds. In this study, we have fused a codon-optimized partial camel lactoferrcin and lactoferrampin DNA sequences in order to construct a fused peptide via a lysine. This chimeric 42-mer peptide consists of complete and partial amino acid sequence of camel lactoferrampin and lactoferricin, respectively. Human embryonic kidney 293 (HEK-293) cells were used for synthesizing this recombinant peptide. Finally, the antibacterial activities of this constructed peptide were investigated under in vitro condition. The result showed that, all construction, cloning and expression processes were successfully performed in HEK-293. One His-tag tail was added to the chimera in order to optimize the isolation and purification processes and also reduce the cost of production. Additionally, His-tag retained the antimicrobial activity of the chimera. The antimicrobial tests showed that the growth rate in the majority of bacterial plant pathogens, including gram negative and positive bacteria, was inhibited by recombinant chimera as the level of MIC values were evaluated between 0.39 and 25.07 μg/ml for different bacterial isolates.

Effects of cLFchimera, a recombinant antimicrobial peptide, on intestinal morphology, microbiota, and gene expression of immune cells and tight junctions in broiler chickens challenged withC. perfringens

“…[42]. Previous findings suggested that cLF36 could attach to the bacterial membrane through electrostatic interactions and physically disrupt bacterial bilayer membranes [12, 13 and 14]. In line with the previous reports [44], the current results demonstrate that AMP can selectively prevent the bacterial growth in the intestine of C. perfringens challenged chickens, which may prove the competitive advantage of cLchimera compared to antibiotics.…”

“…The AMP used in the present study was derived from camel lactoferrin (cLF) consisting of 42 amino acids, which were recently generated in our lab recently (for more details regarding the peptide cLF chimera production, please review previous papers [12, 13 and 14]. Briefly, preparation of recombinant plasmid vector was conducted through transforming recombinant expression vector harboring synthetic cLFchimera into DH5α bacterium [12, 13 and 14]. Next, the latter bacterial colonies were cultured to harvest plasmid extraction.…”

“…More recently, the recombinant form of cLFchimera has been cloned and expressed in E. coli [12] and L. lactic [13] in our lab. The results of in vitro studies showed that cLF36 has antibacterial [12, 13 and 14] antiviral [15], and anticancer [16] properties. Furthermore, the results of an in vivo experiment showed that supplementing E. coli challenged broilers with cLFchimera improved villi morphology in the jejunum, restored microbial balance in the ileum, and improved gene expression of cytokines and tight junctions in the jejunum of challenged broiler chickens [17].…”

Generation of an engineered food-grade Lactococcus lactis strain for production of an antimicrobial peptide: in vitro and in silico evaluation

“…More recently, the recombinant form of cLFchimera has been cloned and expressed in E. coli [64] in our lab. The results of in vitro studies showed that this peptide has antibacterial [62,64,65], antiviral [61], and anticancer [63] properties. Furthermore, the results of an in vivo experiment showed that supplementing E. coli challenged broilers with cLFchimera improved villi morphology in the jejunum, restored microbial balance in the ileum, and improved gene expression of cytokines and tight junctions in the jejunum of challenged birds [14].…”

Antibacterial Activity of cLFchimera and its Synergistic Potential with Antibiotics against Some Foodborne Pathogens Bacteria

“…More recently, a chimeric form of peptide named cLFchimera has been expressed and puri ed in E. coli [5] in our lab. The results of in vitro studies showed that this peptide has antibacterial [5][6][7], antiviral [8], and anticancer [9] properties. Furthermore, the results of an in vivo experiment showed that supplementing E. coli challenged broilers with cLFchimera improved villi morphology in the jejunum, restored microbial balance in the ileum, and improved gene expression of cytokines and tight junctions in the jejunum of challenged birds [10].…”