The Process of Protein Purification - Literature review Example

?INTRODUCTION Recent advancements in molecular biology have led to purification and characterization of proteins. This type of study is important, especially when molecular defects, such as lack of enzymes, or overproduction thereof, are associated with diseases that have seemed incurable in the past. Vaccines, gene therapy and replacement (such as that in insulin-deficiency) all have helped in improving health conditions, and have been based on good elucidation of structures, research on structure-function relationships, and establishment of protein purification specific to the amino acid sequence present. With this in mind, this particular study designed a protocol to purify and characterize the a synthesis of cytochrome oxidase (SCO)-1-like protein 3966 in Streptomyces lividans. As will be seen later, better understanding of SCO proteins is still warranted, as many potential functions of these types of proteins are unclear. Moreover, SCO is a vital enzyme as the cytochrome oxidase c, and in essence the electron chain transport of the mitochondrial respiration mechanism, depends on it. Initial studies of homologues in bacteria have been the usual first step in protein characterization. Many proteins in the eukaryotic cells have been proven to have functional and structural counterparts in bacterial cells. Because of the relative ease of bacterial replication and protein purification, it is thus a method of choice in conducting in depth studies of proteins. REVIEW OF RELATED LITERATURE I. Protein Purification There are factors to consider in doing protein purification. First, aside from proteins, there are other biomolecules present in a cell, such as lipids, carbohydrates, and nucleic acids, all complication the purification process of proteins, Furthermore, a specific protein exists at amounts less than 0.001% of the total protein in a cell (Burgess, 2008). Despite being made of basically the same components, no two proteins are exactly the same. With this we can say that each protein can be purified with a specific set of protein purification protocols that separate the protein of choice not only from the other biomolecules but from the other proteins as well (Burgess, 2008). One of the most common modes of protein purification is column chromatography, whereby the crude solution is passed on to a column of well-compacted silica, alumina, or cellulose. There are many kinds of column chromatography, ion-exchange, affinity, and size exclusion are just some of the more usual protein purification procedures that may be done. Affinity chromatography uses antibodies for a specific protein as part of the column through which the protein solution passes. Although it is highly specific, it is more expensive and much harder to prepare. Size exclusion, on the other hand, depends on the differences of molecular weights of the proteins that are present in the solution. In general, proteins with high molecular weight are eluted fastest as they are not able to get into the small spaces of the column, making their path down the column less impeded. On the other hand, low molecular weight proteins still pass through the tiny spaces within the column, thus slowing down their descent. Although much easier to prepare than an affinity column, a size exclusion chromatography column is less specific, as different proteins of similar weigh are eluted out at the same time, despite them having differences in characteristics, such as the isoelectric point (Burgess, 2008).. For ion exchange chromatography, these beads are charged, thus attracting the oppositely-charged proteins present in the solution to be passed through the column. Depending on the objective of the experiment, the eluent or the bound proteins are collected for further processing such as concentrating. To get the proteins bound on the beads, salt solutions of graded concentrations are passed onto the column. As the concentration of the salt increases, the beads will more likely bind to the salt than to the proteins. Thus, weak ionic proteins are bound weakly to the beads, and are dislodged easily even at lower concentrations of the salt. In contrast, highly ionic proteins are bound strongly to beads, and are only dislodged once highly concentrated salts are used to wash the column (Burgess, 2008).. One of the more commonly used salts for protein purification through column chromatography is ammonium sulfate. This salt is mild and stabilizing to proteins, and is relatively inexpensive, despite it being pure and highly soluble (Burgess, 2008). II. Streptomyces lividans A. Physical characteristics Streptomyces lividans is a highly proliferating species of gram-positive bacteria. They, together with other members of the genus, resemble branching and filamentous fungi, which seem to be twined around one another, thus the name (Greek term, meaning “twisted fungi”). Their close evolutionary association with the eukaryotic fungi is not only evident on their morphological similarities, but in their related life cycles as well (http://microbewiki.kenyon.edu/index.php/Streptomyces, 2010). B. Molecular characteristics The genus is known to have a larger genome and more transcription factors, compared to other prokaryotes. This confers them a more varied metabolic activity and a better resistance against changes in living conditions (http://microbewiki.kenyon.edu/index.php/Streptomyces, 2010). C. Importance in humans Streptomyces are hugely responsible for the production of compost soil used in gardening (http://microbewiki.kenyon.edu/index.php/Streptomyces, 2010). Their interesting molecular characteristics have led molecular biologists to use them in recombinant protein production, successfully translating both prokaryotic and eukaryotic proteins. It serves different advantages to the researchers, such as increased secondary metabolite secretion into the extracellular medium, decreased activity of proteases, its low pathological profile, and it’s established growing conditions (Pimienta et al., 2007). III. Synthesis of cytochrome c oxidase (Sco) proteins A. Molecular Characteristics SOC structure typically contains a thioredoxin fold, which is made up of a central four-stranded ?-pleated sheets (?3, ?4, ?6, ?7) surrounded by three ?-helices (?1, ?3, ?4). On its N-terminal end is a ?-hairpin structure composed of two ?-pleated sheets (?1 and ?2) and a 310-helix (?1). Among eukaryotes, another ?-hairpin structure is present, and is connecting the ?3 and ?6 subunits. This additional structure is exposed to solvents (Figure 1) (Banci et al, 2011). As can be seen in Figure 2, the thioredoxin fold is found to be universal among enzymes of a similar function, as they can also be seen in peroxiredoxins and glutathione peroxidases (Williams et al., 2005). The presence of Cu1+ makes the molecule in a much more stable state. Figure 3 gives a detailed illustration of the conformational changes that happen from an apo- to a holoenzyme. The metal ion attaches the solvent-exposed, ?-hairpin loop to the ?2 helix of the thioredoxin fold. As well, the presence of Ni2+ makes the Cu-SCO complex much more stable (Banci et al., 2011). B. Functions 1. Classical Function: : Promotion of cytochrome c oxidase (COX) production Function of COX In yeasts, it was found to be essential in the accumulation of subunit II of COX. COX, a membrane protein synthesized in mitochondria, and later to be found on the plasma membrane of prokaryotes, is a component of the electron chain transport that utilizes the high energy NADH to power its H+ ion component, creating an electrochemical proton gradient that is used by the adenosine triphosphate (ATP) synthase to produce the high-energy molecule used in most cellular processes (Campbell and Reese, 2002). Mechanism of action The manner by which SCO promotes proper COX assembly is still highly debated, as different mechanisms are found on different organisms, as well as within an organism (Banci et al., 2011). In particular, SCO1 and SCO2 act differently on maturation of COX in humans. Subunit II of COX is a vital part of the enzyme as it makes up the one of two parts of dinuclear copper center that acts as the primary electron acceptor of the protein (Banci et al., 2011). In humans, the two SCO genes, SCO1 and SCO2, code for metallochaperones that are thought to be necessary for the synthesis of COX subunit II. However, Leary et al. (2009) found that the absence of SCO2, but not of SCO1, lead to a reduction in subunit II production. However, newly-synthesized subunit II is better protected in the presence of both SCO1 and SCO2. These suggest that of the two, SCO2-encoded protein is the one necessary for the biogenesis of subunit II. The process by which subunit II in humans is produced is thus hypothesized to be chronologically made up of these steps. First, SCO2 protein interacts with the newly-synthesized subunit II to produce a complex, which in turn attracts the SCO1 and induces the metallation of this metallochaperone by COX17. Finally, the copper is delivered to subunit II, and spontaneously, the SCO proteins are released, giving way to the production of the holoenzyme complex of COX (Leary et al., 2009). 2. Other functions Copper regulation Aside from promoting COX formation, SCO proteins are thought to regulate the copper concentration by distributing the copper in cytosol and intracellular membranes so that all the needs of the basic physiological processes in which copper is needed, such as neurotransmitter biosynthesis, respiration and antioxidation, are met without allowing an increased copper concentration that will allow them to change into its other forms (Cu2+ and Cu3+) that generate harmful radicals. This was demonstrated by Leary et al. (2007) when SCO1- and SCO2-deficient fibroblast cell lines also had low total cellular copper content. The mechanism by which this is achieved by SCO1 and SCO2 is through sending signals from the mitochondria to promote copper retention throughout the cell, instead of to increase the uptake of the metal ion. Redox Signaling The structural similarity of SCO to peroxiredoxin suggests the redox signaling capabilities of SCO. The specificity of SCO to Cu1+ allow itself to act as a switch, whereby presence of oxidants such as hydrogen peroxide converts Cu1+ to Cu2+, which is released from the SCO. The signal is then transferred downstream as vital molecules such as COX are affected by this shift of SCO in a less stable configuration. Evidence that confirms this hypothesis is the observed lethality of hydrogen peroxide on SCO-deficient cells (Williams et al., 2005) C. SCO in prokaryotes SCO has been characterized both in eukaryotic and prokaryotic cells, suggesting its role as a housekeeping protein necessary for aerobic energy production, and as stated above, for other purposes as well. Two prokaryotic homologues, one from Bacillus subtilis and the other from Pseudomonas putida are to be discussed in this chapter. Interestingly, the SCO1-counterpart in P. putida does not have a significant copper-binding activity, unlike those observed from eukaryotic organisms. This means that SCO in this organism does not work to provide materials for COX production. Instead, it acts to reduce metal ions, such as Cu2+ to Cu1+ and Fe3+ to Fe2+. This exclusive redox activity has also been found on other prokaryotic organisms such as Rhodobacter spheiroides. For these bacteria, it was found that the histidine ligand is not surrounded by the hydrophobic residues that are positioned closed together to house the ion. Instead, the SCO homologues of P. putida and R. spheroides have hydrophilic residues that do not stay close with the histidine residue to interact with copper (Figure 4). The position of histidine is thus the definitive factor that determines whether a SCO-homologue acts to charter copper ions or to reduce metal ions (Banci et al., 2011). In contrast, the SCO1 homologue from B. subtilis has a copper chaperone activity. The difference detected is that for the B. subtiliis, the metal-binding motif is exposed to the solvent, while the eukaryotic SCO1 has this motif partially hidden. This suggests that the prokaryotic SCO1 in this case is much more structurally-plastic as compared to the eukaryotic counterpart. This also explains why structural elucidations of B. subtilis SCO1 are usually that of the apo-enzyme (Banci et al., 2011). Figure 5 below is an illustration of the structure of B. subtilis SCO1. References Balatri, E., Banci, L., Bertini, I., Cantini, F., and Cioffi-Bafoni, S. (2003). Solution Structure of Sco1: A Thioredoxin-like Protein Involved in Cytochrome c Oxidase Assembly, Structure, 11, 1431-1443. Banci, L., Bertini, I., Cavallaro, G., and Cioffi-Bafoni, S. (2011). Seeking the determinants of the elusive functions of Sco Proteins, FEBS Journal Burgess, R. R. (2008). Protein Purification [Online] Available at: http://www.wiley-vch.de/books/sample/3527317163_c01.pdf. Accessed: August 18, 2011. Campbell, N. A. and Reece, J. B. (2002) Biology, 6th ed. London: Pearson. Leary, S. C., Sasarman, F., Nishimura, T., and Shoubridge, E. A. (2009). Human SCO2 is required for the synthesis of CO II and as a thiol-disulphide oxidoreductase for SCO1, Human Molecular Genetics, 18(12), 2230-2240. Leary, S. C., Cobine, P. A., Kaufman, B. A., Guercin, G., Mattman, A., Palaty, J., Lockitch, G., Winge, D. R., Rustin, P., Horvath, R., and Shoubridge, E. A. (2007). The Human Cytochrome c Oxidase Assembly Factors SCO1 and SCO2 Have Regulatory Roles in the Maintenance of Cellular Copper Homeostasis, Cell Metabolism, 5, 9-20. Pimienta, E., Ayala, J. C., Rodriguez C., Ramos, A., Mellaert, L. V., Vallin, C., and Anne, J. (2007). Recombinant production of Streptococcus equisimilis streptokinase by Streptomyces lividans, Microbial Cell Factories, 6(20). Williams, J. C., Sue, C., Banting, G.S., Yang, H., Glerum, M., Hendrickson, W. A., and Schon E. A. (2005). Crystal Structure of Human SCO1: IMPLICATIONS FOR REDOX SIGNALING BY A MITOCHONDRIAL CYTOCHROME c OXIDASE “ASSEMBLY” PROTEIN*S, Journal of Biological Chemistry, 280 (15), 15202-15211. (2010). Streptomyces [Online] Available at: http://microbewiki.kenyon.edu/index.php/Streptomyces. (Accessed: August 16, 2011). Read More