Isolation And Purification Of Antimicrobial Peptides

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Antimicrobial peptides which are also called host defence peptides are an evolutionarily conserved components of t innate immune response . These are found among all the stages of the life. The differences which exist between prokaryotic and eukaryotic cells decide the targets for antimicrobial peptides. The antimicrobial peptides are potent, broad spectrum antibiotics which demonstrate potential as novel therapeutic agents. Antimicrobial peptides are likely to kill Gram negative and Gram positive bacteria which includes the strains that are resistant to conventional antibiotics, mycobacteria which includes Mycobacterium tuberculosis, enveloped viruses, fungi and even cancerous cells. Unlike the conventional antibiotics the antimicrobial peptides also have the ability to enhance immunity by functioning such as immunomodulators.

Structure

Antimicrobial peptides are a unique and diversed group of molecules, which are divided into several subgroups on the basis of their amino acid composition and structure. Antimicrobial peptides contain around 12 to 50 amino acids. These peptides have two or more positively charged residues provided by arginine, lysine or, in acidic environments, histidine, and a large proportion hydrophobic residues. The secondary structures of these molecules follow 4 structures, such as

i) α-helical,

ii) β-stranded due to the presence of 2 or more disulfide bonds,

iii) β-hairpin or loop due to the presence of a single disulfide bond and

iv) extended.

Many of these peptides are seen structure less in free solution, and fold into their final configuration upon partitioning into biological membranes. These contain hydrophilic amino acid residues on one side and hydrophobic amino acid residues on the opposite side of a helical molecule. This amphipathicity of the antimicrobial peptides allows to partition into the lipid membrane bilayer. The antimicrobial peptides has a definitive feature of associating with membranes although membrane permeabilisation is not required. These peptides have a variety of antimicrobial activities ranging from membrane permeabilization to action on a range of cytoplasmic targets.

Summary

The host pathogen interaction covers all the concepts of microbial commensalism, colony formation infections and the disease. It is a type of interaction between the virulence capacity of the microorganism and the immune system of the host. When infection is caused the immune system of the host produces antimicrobial peptides against the disease defending the host. Various response levels are occurred due to the host pathogen interaction which depends upon the immune factors of host and virulent factors of pathogen. The immune factors such as cathelicidins, defensins are generated by neutrophils, dendritic cells, macrophages and T-regulatory cells etc. Virulence factors will be of various types based upon the pathogenicity of the pathogen. The antimicrobial proteins are polar molecules which are cationic and are divided into three families such as Defensins, cathelicidins and histatins basing upon the structure, size and the amino acid sequence.

Defensins:

They are small cysteine-rich cationic proteins found in both vertebrates and invertebrates. They act strongly against microbes which include bacteria, fungi and many enveloped and nonenveloped viruses. They contains amino acids ranging from18-45 including 6 to 8 conserved cysteine residues. Cells of the immune system contain these peptides to help in killing phagocytized bacteria, for example in neutrophil granulocytes and almost all epithelial cells. Most defensins function by binding to microbial cell membrane, and, once embedded, forming pore-like membrane defects that allow efflux of essential ions and nutrients.

The genes which are helpful in the production of the defensin are highly polymorphic and rich in arginine residues. The quality of a β-defensin is its small size, high density of cationic charge, and six-cysteine-residues. In general, they are encoded by two-exon genes, where as the first exon encodes for a hydrophobic leader sequence and the second for a peptide containing the cysteine motif.

There are three main known forms of mammalian defensins; α-defensins, β-defensins, and θ-defensins.

In immature marsupials defencins play a great role in the defence against the microbes where as in humans the genome contains θ-defensin genes having a premature stop codon. The more interesting thing here is that the effects of α-defensins on the exotoxins of the anthrax

Cathelicidin

Cathelicidins are one of the types of antimicrobial peptides which belong to a family of polypeptides which are obtained in the lysosomes in the polymorphonuclear leukocytes. Only one cathelicidin is extracted till now from the bone marrow of the human beings. These contain the granules of the myeloid cells and inflamed skin. Cathelicidin peptides are isolated from different species of mammals. Cathelicidins were actually obtained in neutrophils. They are also can be obtained from many other cells namely macrophages, epithelial cells etc

Histatins:

Histatins are the proteins encoded by HTN3, these are called as primary proteins. They contains a, histidine-rich family which is small, salivary proteins, which are encoded by at least two loci namely HTN3 and HTN1. Post-translational proteolytic processing results in many histatins: e.g., histatins 4-6 are derived from histatin 3 by proteolysis. Histatins 1 and 3 are primary products of HIS1(1) and HIS2(1) alleles, respectively. Histatins are believed to have important non-immunological, anti-microbial function in the oral cavity. Histatin and histatin 2 are major wound-closing factors in human saliva. The histatins have both candidacidal and candidastic activities.

Procedure

The procedure is carried out experimentally in two ways such as:

Culturing of the epithelial cells

Isolation, purification and characterization of the antimicrobial peptides from the epithelial cells which are being cultured.

Culturing of the epithelial cells:

Epithelial cells can be cultured on a suitable medium by taking the epithelial cells from the skin of the epithelial layer. Then they are made to infect with suitable pathogen which leads to the stimulation of the host defence against the pathogen. Now these epithelial cell are left aside for the stimulation in a serum free isolate with the clinically isolated bacteria. Then the supernatant is collected and performed next processes like charactering the anti microbial peptides and purifying them.

Isolation, purification and characterization of the antimicrobial peptides from the epithelial cells which are being cultured:

In this method first antimicrobial peptides are isolated from the epithelial cells which are cultured by a process known as cell disruption. There are many techniques namely blending techniques and sonication which includes the lysis of the cell. The next process involves the suspension of the cells which are lysed in the buffer and then the cells are centrifuged to extract the anti microbial peptides and the cell debris is removed away.

Now these extracted antimicrobial peptides are made concentrated by adding salt to it and this salt is made to solvate by adding water and then decreasing the protein salvation present i the solution. By this all the hydrophobic tails are take out and made to get interacted with each other which results in the precipitation of the antimicrobial peptides.

The further step in this process is the usage of the dialysis and gel fibration chromatography for the removal of the salt remains from these peptides. In this method smaller molecules can be separated from the larger molecules by a process called Dialysis in which a semi permeable membrane is used. In the dialysis process salt concentration is added on the both sides of the dialysis bag at a definitive point called equilibrium point. As a result of the addition of salt on the dialysis bag there will be changes in the volumes of salt which leads to decrease in the concentration of the salt in the peptide solution.

Now a process called High Performance Liquid Chromatography (HPLC) by using the antimicrobial peptides purified and characterized earlier. The anti microbial peptides ca be separated by using a variety of liquid chromatography techniques of which four are to be considerd here namely

1)Size exclusion Chromatography

This is one of the simple chromatography technique which is being used for the peptide separation. In this technique peptides are being separated depending upon their size.

2)Normal Phase Chromatography

This is a older technique of the chromatography used in separating the peptides. In this technique an interaction ofadsorbent solute looks over it. The interaction intensity varies with the change in the polarity that means if the polarity is low the the intensity of ineraction will be low and viz versa.

3) Ion Exchange Chromatography

In this technique ionic forces present between the polar mobile phase and the stationary phase play a major role. Here the polar mobile phase is water with small amounts of alcohol or salts where as stationary phase contains definitive sites of acids or bases. In this if salt concentration addition is increased then components of the sample in the column also increases. This method is performed at a fixed pH which creates differences in the charge.

4) Reverse Phase Chromatography

This is the technique where mobile phase will be polar and the stationary phase will be non polar. Majority of the chromatography techniques performed by liquid are done on bonded phases in this method only.

Generally liquid chromatography will be used in the process of purification and the characterization of ati microbial peptides from the junk matter. Chromatography is nothing but a separating technique. The anti microbial peptides obtained here are filtered along with abuffer solution and then passed into a affinity column equilibrated with appropriate buffer. The peptides obtained from this process are bound to column. Now the effluent is slowly added into the column for the elution of the peptides which are bounded to the column. This method is repeated several times to increase efficiacy of column to withhold anti microbial peptides. Now a light beam is used to identify the components present in the eluted materials and this made a note in a chart. By using this method both the quatitative and qualitative information about the anti microbial peptides can be known.

Maldi Technique

Gene Expression Analysis:

The anti microbial peptides also act as the mediators apart from the action of the antibiotics. Because of this the regulation of their gene expression will be strict. Depending upon different stimuli and signalling pathways the expression of the defensins will be varying. As a result they act as effectors molecules. Let us take an example of human β defensin-1 whose expression is found to be constitutive while coming to human β defensin-2 will be not regulated by the infectious and inflammatory stimuli. The expressions of the cathelicidins and the defensins will be unregulated by certain factors namely interleukins, tumours necrosis etc. Epithelial cells will be expressing the receptors which help in detecting the infection and also the inflammation and secretion of anti microbial peptides. Being antibiotic in nature the anti microbial peptides also show some synergistic activity along with lysozyme, lactoferrin etc. Now we can belive that anti microbial peptides will be acting as mediators due to the changes in the expression and gene regulation.

Discussion

The epithelia of primates, insect, plants and some other mammals along with the human epithelial system with a chemical defence system, this has been confirmed by the extraction of anti microbial peptides namely hBD1, hBD2 etc.

The discovery of anti microbial peptides confirms atheory that these peptides belong to innate host immunity which responds strongly against the infection. This laid an clear route for the dovelopment of a new period in the field of antimicrobial theraphy which proves the future for the dovelopment in defending against the infectious diseases which are related to epithelial, gastric or mucosal cell types.

The biological activity of these peptides is not yet know till today which will be considered as one and only disadvantage of these peptides. They are also highly expesive so that they are produced in the little t amounts. Till it has been clear about how the anti microbial peptides are helpful in acting as the mediators of the inflammation in addition to the antibiotics.

Conclusion

Anti microbial peptides will be acting as an main part of the hosts immune system which is called the first lie of the defence. The discovery of these peptides in the fields of biotechnology and pharmacology proved that there is an forward step in treating certain diseases such as skin, respiratory etc. Thus it laid an path for the antimicrobial theraphy dovelopment

The anti microbial peptides can be used as a main source of doveloping drugs in the fields such as biotechnology and related areas like biochemical assyas etc. Even though the derivatives of these peptides are still under process it gives more scope in the future.

The anti microbial peptides will be used in the treatment of the diseases, applied in the field of genome theraphy in coming days. and also used in producing the vaccines. They are also used in the producing the drugs in future where as now these are resitricted because of its biological importance is not yet revealed and also its function.

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