Proteases are important through out the life from conception, birth, life, ageing, to death of all organisms (Abraham and Potter 1989; Lala and Graham 1990). They are biology's version of Swiss army knives, cutting long sequences of amino acids (called peptides) into fragments that fold into functional proteins (Seife 1997). Proteases are interesting molecules that posses harmful and beneficial characters at the same time, they posses catabolic and anabolic characters, they can cause diseases and sever damage to body if inserted from other organisms on one hand but on the other hand some protease from other organisms are used to give a disease relief and healthy life.
A slight change of their balance in body (hypo or hyper activity) can cause disease symptoms. There are almost 50 known human genome sequences in which single mutation can cause the genetically or hereditary disease resulting in the over/ under production of protease or protease inhibitor/ activators, leading to pathological condition (Puente, Sanchez et al. 2003). Proteases are the main focus of modern research to cure several diseases; some of the examples are given in table 1.7.0.
Table 1.7.0: Protease related diseases in Human beings.
Diseases Protease involved Reference
Human Immuno Defficiency
Virus infection HIV-protease (Goldberg and Stricker 1996;
Perryman, Lin et al. 2006) Blood cancer T cell leukemia virus (Li, Laco et al. 2005) Alzheimer's disease amyloid-beta peptides (Beher and Graham 2005) hemostasis, repair, cell survival,
Kawasaki disease PMN-derived elastase, (Saji 2008) Cardiovascular diseases Serine protease Corin (Wu 2007)
Foot and mouth diseases 3C virus protease (Curry, Roque-Rosell et al. 2007) Osteoarthritis diseases proteases (De Nanteuil, Portevin et al.
2001) Aging, hereditary cerebral
hemorrhage, Alzheimer’s down syndromes
Alpha 1-antichmotrypsin (Abraham and Potter 1989)
Air ways injuries proteases (Rennard, Rickard et al. 1991)
Asthma, allergies Bromelain, papain (Baur and Fruhmann 1979) Cancer, breast cancer, apoptosis
irregulations, cancer metastasis
proteases (Hocman 1992; Kennedy 1993;
Das and Mukhopadhyay 1994;
DeClerck and Imren 1994) (Rochefort, Capony et al. 1990;
Rochefort and Liaudet-Coopman 1999)
According to a careful estimation of Southan, C. protease comprises ~1.8% of the human genome, and genome data annotation revels protease inhibitor ratio as 10:1 (Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.). This estimation ended with 700- 1100 proteases and 70–110 protease inhibitors. Protease comprises little higher almost 5% of genomes of infectious organisms (Southan 2000).
In most of the pathogenic organisms, proteases are the major causes of pathogenesis (Travis, Potempa et al. 1995). It severity range from mild fever, or pain to even death, like in case of cane disease caused by Clostridium botulinum. When pathogens invade there is a sensation of pain in the result of dysregulation of kallikrein and kinin pathway (figure: 1.7.0.) caused by bacterial protease (Nilsson, Carlsson et al. 1985;
Maeda and Molla 1989).
Figure 1.7.0: Functions of bacterial proteinase in infection Source: (Miyagawa, Nishino et al. 1991)
They mainly act as degradative enzymes among the lower to higher organisms and in some instance they act very specifically and help the cell in the vital function of protein folding, cell signaling, hormonal communication etc. therefore deficiencies of theses enzymes in biological system can convert into diseases. Concerning these biological effects of protease deficiencies, enzyme therapy for protease is in practice.
Numbers of orally administrable protease drinks prepared from natural sources are available at the market. Several reasons have been describing in this respect
(http://www.enzymeessentials.com/HTML/protease.html; http://www.enzymeresearchgroup.net/protocols.php). It has been described that proteases, when taken orally can be absorbed by alpha2 macroglobulins. They used to encounters dead, damage and foreign unidentified protein particles of allergens and pathogenic factors from bacteria, fungi, insects and other organisms.
1.8.0. Introduction to the organism – Aliivibrio salmonicida:
A. salmonicida LFI1238 is a halophilic (“salt loving”) and psychrophilic (“cold loving”), curved, gram-negative bacterium (Hoff 1989; Colquhoun and Sorum 2001).
It is known as the causative agent of “cold-water vibriosis (CV)” or “Hitra disease”, occuring at low temperature and causes hemolysis and tissue degradation in fishes (Salte, Nafstad et al. 1987; O'Halloran 1993; Stephen 1993). It was predominant in winter time with low water-temperatures (Holm and Jørgensen 1987). In contrast to other septicaemic, hemorrhagic, pathogenic bacteria, no exotoxin have been identified until now (Hjeltnes, Andersen et al. 1987; Holm and Jørgensen 1987). For this reason A. salmonicida is an interesting model organism for the study of temperature and host adaptation mechanism.
Figure 1.8.A: Electron Microscopic photograph of Aliivibrio salmonicida (Photo taken by Steinar Paulsen, Protein Research Group, UITø, Norway)
Psychrophiles have the ability to survive and proliferate at low temperatures.
They have been modified under the constant cold environments challenges. These organisms and their building blocks (proteins) posses the quality that distinguish them from the organism that can not survive in cold environment. D’Amico et al. described some of these challenges like reduced enzyme activity, decreased membrane fluidity, altered transport of nutrients and waste products, decreased rates of transcription, translation and cell division, protein cold-denaturation, inappropriate protein folding, and intracellular ice formation. Cold-adapted organisms have successfully evolved features, genotypic and/or phenotypic, to surmount the negative effects of low temperatures and to enable growth in these extreme environments (D'Amico, Collins et al. 2006).
To study the genome of A. salmonicida a project was organized, by department of molecular biotechnology, UiTø and NorStruct. The genome consists of two chromosomes, two megaplasmids and four plasmids. Shot gun libraries have been constructed in collaboration with, the Welcome Trust Sanger Institute, to sequence the whole genome, (www.sanger.ac.uk/Projects/V_salmonicida/). This genome have been recently sequenced and published (Hjerde, Lorentzen et al. 2008). (Figure: 1.8.B)
Figure 1.8.B: chromosomal circular diagrams (outside to inside) ): scale (in Mb), unique CDSs compared to the other Vibrionaceae species (red), orthologues shared with the other Vibrionaceae species (green), IS element transposases (purple), dark blue, pathogenicity/adaptation; black, energy metabolism; red, information transfer; dark green, surface associated; cyan, degradation of large molecules; magenta, degradation of small molecules; yellow, central/intermediary metabolism; pale green, unknown; pale blue, regulators; orange, conserved hypothetical; brown, pseudogenes; pink, phage + IS elements; grey, miscellaneous. The positions of phage elements and GIs larger than 5 kb are marked (red); source: (Hjerde, Lorentzen et al. 2008)
1.9.0. Protein expression through cloning:
Proteins in host organisms express in a limited quantity and usually in the response of certain stimuli. The main objective of the cloning is to obtain the elevated level of target protein expression, higher to the source organism of target protein, so that it can be obtains in milligram quantities necessary for structural and functional characterization. Cloning systems can be utilized in a controllable manner to expresses the target protein in response to the stimulants, when ever needed. These systems require the inducer or stress condition to unblock the progressive transcription of cloned gene of target protein.
With the development of cloning and protein expression technology, numbers of choices are present not only for cloning methodology but also for cloning systems that composed of expression vector and expression clone (choice of cell types for expression). Careful selection of cloning system according to the protein characteristics and tag requirement for protein purification is the major and initial step in protein expression through cloning technology. The second scaling up step is the optimization of growth conditions or media constituents for highest possible soluble yield in a single batch. As a whole, theoretical and experimental decision in these two steps govern the economically feasible expression system development.
1.9.1. Bioinformatic analysis of protein:
Before trying to express a target protein a general bioinformatics analysis of protein is required to develop a strategy for purification for example PI calculation in case of ion exchange chromatography. A model building is also helpful to guess the exposed terminal for tag attachment (N/C-terminal tag) for purification purpose.
Closely related proteins information is also useful to design a cloning/ purification strategy and possibility of heteromer formation. Some proteins are designed in nature for extra-cellular or periplasmic expression, these protein can be detected by analysis through SignalP (Bendtsen, Kiemer et al. 2005; Emanuelsson, Brunak et al. 2007).
Such proteins usually express when transported out of the cell or in periplasm.
Such target proteins usually exported out with the help of the signal sequence from
the expression vector and cloned without signal sequence. Some proteins are also expressed in zymogene form, in that case pro sequence needed to be identified by aligning with an enzyme of same category, when needed to be express in the active form. These proteins by understand the Half life, THMMH, solubility
1.9.2. Primer designing:
Primers should be design in such a way that it will bring the gene code of target protein in frame with the tag and initiation codon. In spite of all the precautions possibility of missing frame or any mutation during the purification from the gel (by excess UV exposure) can be indicated with the sequencing of cloned gene.
1.9.3. Selection of cloning technology:
With recent advancement in Cloning science, traditional cDNA cloning method is now replacing several types of high throughput cloning methods. Numbers of factors are important in their selection that includes, high fidelity (assurance), ease of use, reliability of system, validation of correctly cloned system, flexibility to change the express species or vector and overall cost of recombination, time consumption (Marsischky and LaBaer 2004).