Introduction
Proteasome
Protein synthesis and protein degradation are two universal complementary processes, permanently occurring in living cells; both play an important role in maintaining biological homeostasis and regulation of different intracellular processes.
Particular attention needs the regulated degradation of specific proteins, whose machinery dysfunction can lead to aberrant expression of proteins and consequent deleterious effects for the cell or the organism (Borissenko and Groll 2007).
Before the pioneer Etlinger and Goldberg’s work in 1977 (Etlinger and Goldberg 1977), protein degradation (proteolysis) in cells was thought to rely mainly on lysosomes, membrane-bound organelles containing acids and cathepsins, cysteine or aspartate family proteases. These are able to degrade and then recycle exogenous proteins and aged or damaged organelles (Lodish 2004).
Etlinger and Goldberg work on ATP-dependent protein degradation in reticulocytes, which lack lysosomes, suggested the presence of a second intracellular degradation mechanism.
Now it is known that proteolysis occurs in different cellular compartments: cytosol, lysosome and endoplasmic reticulum. In addition, regulatory intramembrane proteolysis is emerging as an important player in several critical processes as cellular differentiation, lipid metabolism and response to unfolded proteins. However, the mechanism suggested by Etlinger and Goldberg, the ubiquitin-proteasome system (UPS) remains the major component of the non-lysosomal protein degradation pathway and its main character, the proteasome, is involved in a variety of essential biological processes, as well as protein quality control, antigen
ubiquitin is not present, but proteolysis is still ATP-dependent, as in the UPS pathway (Goldberg 2005).
Proteasome structure
The proteasome subcomponents are often referred to by their Svedberg sedimentation coefficient (denoted S).
The constitutive proteasome is known as the 26S proteasome, a multifunctional, 2,500 kilodaltons (kDa) molecular machine, in which several enzymatic (proteolytic, ATPase, de-ubiquitinating) activities function together with the ultimate goal of protein degradation (Borissenko and Groll 2007).
In eukaryotes, 26S proteasomes are composed of the cylinder-shaped multimeric protein complex referred to as the 20S proteasome core particle, capped at each end by the regulatory component termed the 19S complex or PA700 (Hough et al. 1987). The other proteasome type presents in all animal having an immune system, is known as immunoproteasome; it is composed by the 20S core associated with the 11S regulatory complex or PA28.
Immunoproteasome plays a critical role in the function of the adaptive immune system. Peptide-antigens are displayed by the major histocompatibility complex (MHC) class I proteins on the surface of antigen-presenting cells. These peptides are products of proteasomal degradation of proteins originated by the invading pathogen. The immunoproteasome is induced by interferon gamma (γ) during the immune response and is able to produce peptides of the optimal size and composition for MHC binding. γ-interferon acts on genes coding for the regulatory complex PA28 and specialized catalytic subunits called β1i (LMP2), β2i (MECL) and β5i (LMP7) with altered substrate specificity in respect to their constitutive variants β1, β2, and β5.
Another β5 variant subunit, β5t, is expressed exclusively in cortical thymic epithelial cells, which are responsible for the positive selection of developing thymocytes. This variant leads to a thymus-specific "thymoproteasome" whose is supposed to be involved in generating the MHC class I-restricted CD8(+) T cell repertoire during thymic selection (Murata et al. 2007).
20S core particle
20S particle consists of four stacked heptameric ring structures forming a hollow cylinder. Rings are composed of two different types of subunits; α subunits have structural function, whereas β subunits are predominantly catalytic.
The outer two rings in the stack consist of seven α subunits each; they serve as docking domains for the regulatory particles and their N-termini form a gate that blocks unregulated access of substrates to the interior cavity (Smith et al. 2007). The inner two rings each consist of seven β subunits and contain the protease active sites that perform proteolysis reactions.
All 14 different eukaryotic proteasomal subunits contain characteristic insertion segments and termini, which represent well-defined contact sites between related subunits and cause their unique location at special positions within the particle. Electron micrographs of 20S proteasomes revealed its molecular dimensions to be ~160 Å (angstroms) in length and ~120 Å in diameter (Borissenko and Groll 2007). The interior chamber is at most 53 Å wide, though the entrance can be as narrow as 13 Å, suggesting that substrate proteins must be at least partially unfolded to enter (Nandi et al. 2006).
The detailed composition of subunits was first elucidated by crystal structure analysis of the archaebacterial proteasome from Thermoplasma acidophilum (Lowe et al. 1995) and subsequently in a variety of organisms, animals, archaea and eubacteria (Groll et al. 2003; Hu et al. 2006; Kwon et al. 2004).
Structural data show that size and shape of 20S proteasome is relatively conserved even if the number and diversity of subunits contained in the 20S core particle depends on the organism. Though the eukaryotic primary sequences of α- and β-subunits are quite different, they show similar folding, which is also found in the subunits of HslV, the proteasomal analogue of eubacteria. This common folding
However, in archeabacteria all α and all β subunits are identical, while eukaryotic proteasomes contain seven distinct types of each subunit (Fig 1).
The α subunits are more conserved than the β subunits and form a selective barrier between the catalytic chamber and the cytoplasm. They are the sites for the binding of regulatory particles, as PA28 and PA700, and for entry and exit of substrates. On the other hand, β subunits harbour the catalytic site. In mammals, the catalytic functions are held only by the β1, β2, and β5 subunits; although they share a common mechanism, they have three distinct substrate specificities, considered chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide-hydrolyzing (PHGH).
Fig1. Comparison of the subunit composition of 20S proteasomes from different organisms. The archaebacterium T. acidophilum proteasome contains single α and β proteasome subunits while, yeast proteasomes are composed of seven different α and β subunits. In mammals, three constitutive proteasomal β subunits, β1, β2 and β5, are replaced by β1i, β2i, and β5i, which are induced in response to inflammatory signals, e.g. IFNγ. From (Nandi et al. 2006).
PA700 regulatory complex
The 26S constitutive proteasome consists of the 20S proteasome core, bound to a 19S cap also known as PA700 (Fig.2 and 3).
20S proteasome is able to degrade unfolded or loosely folded proteins and peptides in an ATP-independent manner; however, it cannot degrade ubiquitin-protein conjugates because its proteolytic active sites are sequestered within the lumen of this cylindrical complex, to avoid non-specific degradation of folded cellular proteins.
In presence of ATP, the 19S complex becomes associated with each end of the 20S cylinder, to form the completely functioning 26S proteasome (Smith et al. 2007). The eukaryotic 19S particle consists of 19 individual proteins and is divisible into two subassemblies, a 10-protein base that binds directly to the outer α rings of the 20S core particle, and a 9-protein lid where polyubiquitin is bound.
Six of the ten base proteins are ATPase subunits and are evolutionary homolog of ATPase existing in archaea, called Proteasome Activating Nucleotidase, PAN, which regulates 20S function in many archeabacteria (Zwickl et al. 1999).
The 19S regulatory ATPase particle is responsible for stimulating the 20S to degrade proteins, allowing proteins to enter the proteasome channel and oligopeptides to exit. Its primary function is to open the gate in the 20S that blocks the entry of substrates into the degradation chamber (Kohler et al. 2001). The mechanism by which the proteasomal ATPase open this gate has been recently elucidated (Smith et al. 2007). 20S gate opening, and thus substrate degradation, requires the C-termini of the proteasomal ATPases, which contains a specific motif, HbYX. The 19S ATPases C-termini bind into pockets between neighbouring α subunits according the "Key-in-a-lock" model: C-termini interact with conserved residues in the pocket, inducing rotation in the α subunits and displacement of a reverse-turn loop that open the gate and stabilizes this open conformation (Rabl et al. 2008; Smith et al. 2007).
Fig2. Schematic of the 20S core protease (CP) and the 26S proteasome. The 26S proteasome is composed of the barrel-shaped 20S complex with a molecular weight of about 700 kDa capped by two 19S regulatory particles (RP). The molecular weight for this 26S proteasome is about 2000 kDa. The 19S regulatory particle recognizes the polyubiquitin tag on targeted substrates and unfolds the substrate to enter the proteolytic chamber. The 20S core particle contains the catalytic sites responsible for the proteolysis. (Wang et al. 2006).
Fig.3: Surface representation of a cut-open view of the 20S proteasome. In blue is indicated the 19S complex, in yellow the 20S core, with its alfa and beta rings. The active sites are shown as red spheres.
Noteworthy, the gate-opening does not require ATP hydrolysis energy. This is only used for substrate unfolding process, while ATP-binding is sufficient for triggering of the other steps, as complex assembly, translocation and proteolysis (Smith et al. 2005).
19S regulatory particles are believed to assemble by union of their two separately assembled subcomponents, the ATPase-containing base and the ubiquitin-recognizing lid. The six ATPases in the base may assemble in a pair wise manner mediated by coiled-coil interactions. The order in which the nineteen subunits of the regulatory particle are bound is a likely regulatory mechanism that prevents exposure of the active site before assembly is complete (Sharon et al. 2006).
PA28 regulatory particle
20S proteasomes can also associate with a second type of regulatory particle, the 11S regulatory particle (PA28), a heptameric structure that does not contain any ATPases and can promote the degradation of short peptides, but not of complete proteins. It is presumed that this is because the complex cannot unfold larger substrates.
The mechanisms by which it binds to the core particle through the C-terminal tails of its subunits and induces α-ring conformational changes to open the 20S gate suggest a similar mechanism for the 19S particle (Forster et al. 2005).
The expression of the 11S particle is induced by interferon gamma and is responsible, in conjunction with the immunoproteasome β subunits, for the generation of peptides that bind to the major histocompatibility complex MHC class I (Wang and Maldonado 2006).
20S Assembly
followed by cleavage of the N-terminal prosequences in the β subunits, likely by autocatalysis, activating them (Borissenko and Groll 2007).
α subunits result able to form a ring by themselves, while β subunits require preformed α rings as template for their assembly.
The processing of the β subunit occurs after the assembly of complete proteasome complex to prevent non-specific hydrolysis of cellular proteins (Zwickl et al 1994). This processing exposing the N-terminal active site threonine is dependent on the presence of the catalytic Thr-1 and Lys-33 and is mediated mainly by salt bridges and hydrophobic interactions between conserved alpha helices (Witt et al. 2006). Only three β subunits undergo processing of propeptides after assembly in eukaryotes that, hence, present only three active sites per β ring, unlike the T.acidophilum proteasome which harbours seven active sites per ring.
The incorporation of one β facilitates the incorporation of another β as the localization of different subunits within the proteasome ring is fixed.
Mammalian proteasomes display displacement of the active β subunits, β1, β2 and β5 by interferon (IFN)-γ inducible subunits β1i (LMP2), β2i (MECL) and β5i (LMP7) respectively. A model has been proposed to explain the preferential incorporation of these IFNγ-inducible subunits into immunoproteasomes. Here β1i and β2i are incorporated in ‘early’ proteasomes and the incorporation of β5i results in formation of active immunoproteasomes (Nandi et al. 2006).
Propeptides play multiple roles and support the proper folding and assembly of β subunits; in addition, they protect the N-terminal threonine from acetylation mediated inhibition.
Studies have also demonstrated a role of ubiquitin mediated proteolysis-1 (UMP1) protein, for proper proteasome maturation in S. cerevisiae. Remarkably, Ump1p binds to precursor proteasomes and gets degraded by the same proteasome after maturation (Ramos et al 1998).
Homologues of UMP1 have been identified in humans, where are known as hUMP1 or the proteasome maturation protein (POMP). POMP has been shown to be up-regulated in cells treated with IFNγ or proteasome inhibitors. In fact, it has been observed that proteasomal inhibition leads to increased expression of proteasomal genes, facilitating formation of new functional proteasomes to compensate for the
loss (Meiners et al. 2003). POMP is induced by IFNγ and binds to β5i (LMP7) more efficiently than β5, resulting in accelerated formation of immunoproteasomes. In fact, reduced POMP expression results in lower formation of proteasomes, reduced MHC class I and induction of apoptosis (Nandi et al. 2006).
Proteolysis
Eukaryotic proteasomes display three major peptidase activities, based on cleavage of fluorogenic peptides that can be easily assayed: chymotrypsin-like activity (cleavage after hydrophobic amino acids), trypsin-like activity (cleavage after the basic amino acids) and caspase-like activity (cleavage after acidic amino acids). In addition, two other activities, cleavage after branched chain amino acids (BrAAP) and small neutral amino acids (SNAAP) are also known. The three major activities of yeast 20S proteasomes, caspase-like, trypsin-like and chymotrypsin-like activities can be correlated directly to the three subunits β1, β2 and β5, respectively, as demonstrated by mutation studies (Groll et al. 1999).
The mechanism of proteolysis by these β subunits is through a threonine-dependent nucleophilic attack. The surrounding Lys and Arg residues contribute to lowering the electrostatic potential of the γ-oxygen of active Thr. Close to Thr, there are two Ser residues and one Asp, which are required for the conformational stability of Thr. A cluster of water molecule is close to Thr and presumably plays a major role in deprotonation of the reactive threonine hydroxyl.
Generally, the maximum likelihood of substrate cleavage depends on its mean residence time at the proteolytically active sites, so that the product cleavage pattern is directly related to the affinity of substrates for the individual binding clefts.
NF-κB, are synthesized as inactive precursors whose ubiquitination and subsequent proteasomal degradation converts them to an active form. Such activity requires the proteasome to cleave the substrate protein internally rather than processively degrading it from one terminus. It has been suggested that long loops on the surface of these proteins serve as the proteasomal substrates and enter the central cavity, while the majority of the protein remains outside. Similar effects have been observed in yeast proteins; this mechanism of selective degradation is known as regulated ubiquitin/proteasome dependent processing (RUP) (Rape and Jentsch 2004).
The ubiquitin-proteasome pathway (UPS)
In eukaryotes, the ubiquitin-proteasome pathway (UPS) is a strictly controlled complex enzymatic process starting with protein tagging by ubiquitin for recognition and further degradation by 26S proteasome, the eukaryotes constitutive form of proteasome.
Proteins target for degradation depends on multiple factors: the presence of specific sequences (e.g. the destruction box in cyclins) or the amino-terminal residue may be important, for e.g. proteins with a basic amino acid at the amino-terminus are less stable compared to those containing other amino acids, according to the so called “N-end rule” (Nandi et al. 2006). In addition, aged proteins may display a hydrophobic patch due to denaturation that are recognized and routed for degradation. More importantly, cellular signalling events target proteins by post translation modifications (e.g. phosphorylation or oxidation) that undergo degradation. This selective protein degradation plays crucial roles during cellular decision making and renders this process extremely important (Varshavsky 2005). Ubiquitin is a 76-residue polypeptide so named due to its ubiquitous nature. It is
highly conserved among eukaryotes but absent from archaea and bacteria, even if in
E. coli a C-terminal marker peptide of 11 amino acids, named SsrA, was found to mark proteins for degradation, suggesting the presence in bacteria of proteolysis mechanism similar to the UPS (Karzai et al. 2000).
Ubiquitin is covalently bound to the target protein by an isopeptide linkage between its carboxy terminal glycine and the ε-amino group of lysine in the protein (Fig 4). In some proteins (for e.g. the lacking lysine ones), poly-ubiquitination may occur at the amino terminal residue (Ciechanover and Ben-Saadon 2004). Similar isopeptide linkage is formed between the carboxy terminus of ubiquitin with the ε-amino group of lysine of another ubiquitin molecule to form poly-ubiquitin chains.
Fig.4 Ubiquitin-protein isopeptide bond. The ubiquitin carboxy terminal glycine binds the ε-amino group of lysine in the target protein by a bond similar to the peptide one and therefore called “isopeptide bond”.
The lysine residue on which poly-ubiquitination occurs is important because mono-ubiquitination of proteins could have other functions, e.g. endocytosis, histone regulation, virus budding etc. (Hicke 2001).
al. 2006). The E1-ubiquitin thiol ester is recognized by the ubiquitin-conjugating enzyme (E2) to which ubiquitin is transferred by another thiol ester linkage. All E2 enzymes harbour a conserved core that is utilized in binding the ubiquitin ligase (E3). Each E2 can bind more than one E3. Importantly, E3 enzymes are responsible for the final target selection and specificity because they recognize the protein to be ubiquitinated and catalyze the transfer of ubiquitin from E2 to this target protein. A target protein must be labelled with at least four ubiquitin monomers (in the form of a polyubiquitin chain) before it is recognized by the proteasome lid (Thrower et al. 2000). Conventional ubiquitination enzymes E1, E2, and E3 add only limited number of ubiquitin moieties, so, in some cases, it is necessary the additional activity of certain ubiquitin-chain elongation factors that catalyse multi-ubiquitin chain assembly in collaboration with E1, E2 and E3. These enzymes have been proposed to be termed ‘E4 enzymes (Hoppe 2005).
The number of E1, E2, and E3 proteins expressed depends on the organism and cell type, but there are many different E3 enzymes present in humans, indicating that there is a huge number of targets for the ubiquitin proteasome system.
The action of the ubiquitinating enzymes is countered by that of de-ubiquitinating enzymes, or isopeptidases, which are able to remove ubiquitin from poly-ubiquitinated substrates. De-ubiquitination is needed before proteins entering in the proteasome but it might also be important in reversing the ubiquitination of specific proteins and, thus, in preventing, possibly transiently, their degradation.
After a protein has been ubiquitinated, it is recognized by the 19S regulatory particle of the 26S constitutive proteasome in an ATP-dependent binding step (Liu et al. 2006). Substrate protein must then enter into the 20S proteasome core to come in contact with the proteolytic active sites. Because the 20S particle central channel is narrow and gated by the N-terminal tails of the α subunits, the substrates must be de-ubiquitinated and least partially unfolded before they enter the core. The passage of the unfolded substrate into the core is called “translocation”.
The ATP molecules bound before the initial recognition step are hydrolyzed before translocation. Energy from ATP hydrolysis is used for substrate unfolding (Smith et al. 2005). Passage of the unfolded substrate through the opened gate occurs via facilitated diffusion if the 19S cap is in the ATP-bound state (Smith et al. 2007).
The mechanism for unfolding of globular proteins is general, but it was seen (Zhang and Coffino 2004) that some amino acids sequences can decrease the efficiency of proteasomal degradation by inhibiting the protein unfolding: certain Epstein-Barr virus gene products bearing a glycine and alanine reach sequence can stall the proteasome, helping the virus propagate by preventing antigen presentation on the major histocompatibility complex.
Into the proteolytic chambers present in the 20S, the peptide bonds are attached and about 8 or 15 oligopeptides are released. These released oligopeptides could be transported into the endoplasmic reticulum and bind to MHC (Major Histocompatibility Complex) class I molecules for antigen presentation in immunosystem cells or could be cut by cytosol peptidases, as well as endopeptidases, aminopeptidases and carboxypeptidases and the resulting amino acids recycled for further uses.
Fig.5. Protein ubiquitination pathway. Ubiquitin (Ub) is activated by E1 and transferred to the E2 enzyme and is, finally, conjugated to substrate proteins with a specific E3 ligase. Further polyubiquitination is required to target proteins for degradation (Nandi et al. 2006).
Proteasome in Aging
Aging is due to the gradual accumulation of unrepaired random molecular faults, which leads to an increased fraction of damaged cells and eventually to the functional impairment of older tissues and organs.
Cellular components are continuously renewed by the processes of catabolism and re-synthesis as they wear out or become damaged. However the mechanisms of renewal are not perfectly efficient and over time there may be an increase in damaged molecular structures.
Accumulation of damaged proteins is dependent on an increased occurrence of damage to proteins but also on a decreased elimination of modified protein, as suggested by the Terman the "garbage catastrophe theory" of ageing (Terman 2001). Terman claims that the process of ageing may derive from imperfect clearance of oxidatively damaged, relatively indigestible material, the accumulation of which further hinders cellular catabolic and anabolic functions (Fg6).
It has been observed that there is a progressive decline in the overall proteolytic capacity of the cell with age (Bulteau et al. 2000; Szweda et al. 2002) resulting in an accumulation of oxidized and cross-linked proteins (Sitte et al. 2000) which can form pathogenic aggregates into the cells (Chondrogianni and Gonos 2005). Particular attention was posed on decrease in the activity of the UPS with age and was seen that this occurs in several tissues, although, does not seem to be universal (Carrard et al. 2002; Ferrington et al. 2005; Keller et al. 2004; Ward 2002).
Analysing the different steps in UPS degradation, it was discovered that qualitative rather than quantitative changes set the basis for UPS malfunctioning in aging and that UPS ubiquitination step does not seem to be particularly affected by age.
Consequently, the accumulation of Ub-conjugated substrates, common in most aged tissues and in different age-related disorders, is likely to result from a decrease in their efficient removal by the proteasome (Carrard et al., 2002).
Fig6. Intracellular fate of altered proteins. In normally functioning cells (Young) damaged proteins are recognized and target for degradation by the intracellular proteolytic systems. As cells age (Old) the defective activity of the major proteolytic systems leads the intracellular accumulation of damaged/unfolded protein products into aggregates. Protein aggregates slowly accumulate in all cells through the life, but their formation can be precipitate under particular cellular conditions or in certain pathologies (Martinez-Vicente et al. 2005)
Fig.7 UPS system and its changes in aging. Two major steps, ubiquitination (left) and degradation (right), contribute to the removal of cytosolic proteins by the UPS. Already identified age-related changes in the UPS (green callouts) and other possible changes that could explain the defective UPS activity in aging (yellow callouts) are shown. Abbreviations: Ub, ubiquitin; E1, ub-activating enzyme; E2, ub-conjugating enzyme; E3, ub-ligase; E4, ub-elongating factor; CP, catalytic proteasome; RP, regulatory proteasome (Martinez-Vicente et al. 2005).
Therefore it is now accepted that the proteolytic ability of the proteasome is modulated in vivo by multiple factors, and that age-dependent modifications in these factors are probably responsible for altered proteasome activity (Carrard et al. 2002; Ferrington et al. 2005). In fact, a recent study in aged muscle has revealed an increase in the content of the 20S proteasome with age (mainly due to increase in immunoproteasome), concomitant with a severe decrease in the content of regulatory proteins (Ferrington et al., 2005). This deficit of the regulatory subunits is responsible for the inadequate activation of the 20S proteasome with age.
Failure of proteasome function with age could be due to changes in the protease (decreased proteasome expression; alterations and/or replacement of proteasome subunits), changes in the modulator molecules (endogenous regulatory subunits and their partners) and changes in the proteasome substrates (decreased proteolytic susceptibility, cross-linking of proteins, etc) (Fig.7).
Changes in proteasome structure have been widely studied and a shift between catalytic β1, β2 and β5 subunits in favour to their inducible isoforms β1i, β2i and β5i together with post-transcriptional modification of these subunits was noted (Bardag-Gorce et al. 1999; Bulteau et al. 2000; Carrard et al. 2003; Ethen et al. 2007; Husom 2003; Vernace et al. 2007).
Aging modification of proteasome structure and function needs more studies; this is the aim of the European project “Proteomage” that try to shed new light on aging and age-related disease mechanisms.
This work, taking part of the Proteomage project, was carried out in the Phage Display Group Laboratories in the Department of Molecular Biology, University of Aarhus, Denmark, under the supervision of professor Peter Kristensen.
It is focused to the selection of antibodies against proteasome subunits β1, β1i, β7 and α4 using phage display technology.
Phage Display
Human disease as well as aging are often caused by or associated with alteration of protein expression and function. To evidence these alterations it is important to be able to analyze and characterize a large number of genes and genes products.
The understanding of any biological process including progressive loss of function that occurs in aging is based on a variety of data obtained from different sources. Genetic information is useful to understand where and when a gene is activated or inactivated. Therefore, there is another aspect to consider: post-transcriptional regulation, the sum of all steps ranking from DNA translation to the protein activity. This include the stability and half-life of mRNA, the rate of translation of individual mRNA in protein, post translational modifications, the driving of protein in its subcellular place of action and the rate of protein degradation. All these steps can be altered in aged or diseased cells and can influence the protein function.
Following the introduction of polymerase chain reaction (PCR) a number of techniques have become available capable of analysing complex biological systems in a high-throughput manner as well as differential display, serial analysis of gene expression, DNA chip technologies.
Aging and many complex diseases are not influenced by only one gene or its product but by interaction of different factors with a great influence on cell state (Goyns et al. 1998; Lee et al. 1999; Rattan 1995). Thus, even small changes in their levels can be important for aging or disease progression.
After the identification of these factors, it is important to monitor their presence, function and localization in the cell. Therefore, it is useful have a method allowing to see specific proteins and to evaluate their presence into the cell.
Antibodies (Abs) are the natural answer to this question.
Antibodies are gamma globulin proteins used by vertebrate immune system to recognize and to fight unknown and potentially-pathogen molecules entering the body. Therefore, antibodies (Abs) are constructed to have a general structure very similar for all Abs and a small region at the tip of the protein extremely variable. This region, called hypervariable region, allows millions of antibodies with slightly different tip structures to exist.
Each of these variants can bind to a different target, known as an antigene, giving rise to an overall huge amount of antibodies that allows the immune system to recognize an equally wide diversity of antigens. The unique part of the antigen recognized by an antibody is called an epitope. Epitopes bind with their antibody in a highly specific interaction that allows antibodies to identify and bind only their unique antigen in the midst of the millions of different molecules making up an organism.
For their features, Abs had been widely used in biology since long time, mainly in immuno-detection systems. To obtain specific Abs the labour intensive and time spending method of hybridoma technology has often been used. After introduction of phage display technology there was another speed, cheap and easy way to isolate a single antibody from a huge population.
Advantages of using phage display instead of hybridoma technologies are multiple: phage display does not require animals for production of Abs, but only bacteria, easier and faster to handle and a large amount of work in phage display is in vitro, it allows to manipulate the selection and to introduce modification to the gene by genetic engineering technologies. Further, there were been developed phage display systems using antibody fragments instead of the entire Abs. These fragments (as Fv, scFv or Fab), as seen below, are small, so they can be added to the phage without too much altering the phage structure itself; however they present the same high variability and versatility of antibodies, because of they are made from their own hypervariable domains.
Technique and concept
In 1985 Smith and colleagues (Smith 1985) established a method for presenting polypeptides on the surface of filamentous phage, a virus that infects Escherichia coli. In contrast to other phages (e.g., T4), filamentous phages (e.g., fd or M13) are specific bacteriophages that replicate and assemble without killing the host cell.
This technique was originally developed to map epitope-binding sites of antibodies by panning random peptide-phage libraries (big amount of phages each one displaying a different peptide) on immobilized immunoglobulins. Since the pioneering work of Smith, phage display technology based on these phages has grown becoming a widely used technique with application in a lot of different systems and uses for selecting peptides and proteins with desired functions and properties from molecular libraries. For general view (Schimmele et al. 2005; Sergeeva et al. 2006)some examples of phage display application can be found here: (Brissette et al. 2006; Kelly et al. 2006; Kolonin et al. 2006; Rajotte et al. 1998; Rajotte and Ruoslahti 1999).
The principle underlying all phage display systems is the physical linkage of a polypeptide's phenotype to its corresponding genotype (Paschke 2006).
In practice, the protein or peptide to be displayed is fused together with one protein of the phage coat. Such fusion protein is located on the phage external surface and the wanted protein or peptide is exposed (displayed). The phage can be used to bring this protein in contact with a specific receptor and to evaluate their ability for binding.
The genetic information encoding the displayed fusion protein is inserted into the phage genome and therefore packaged together with it inside the same phage particle. Hence, the genotype–phenotype coupling occurs before the phages are released into the extracellular environment, ensuring that phages produced from the same bacteria cell clone are identical.
Usually huge phage library are created, each phage displaying a different protein or peptide, then these libraries are used to detect, with one or more round of selection and amplification in Escherichia coli, the protein/peptide that binds the receptor.
The M13 phage
Structure
Fig.8a Representation of M13 virion, (Khalil et al. 2007)
Fig.8b Schematic representation of M13 structure with coat protein localization, from (Paschke 2006)
Since 1985 and the pioneer work of Smith, there are been created many display systems as well as RNA display,(Ja et al. 2005), ribosome display (Lipovsek and Pluckthun 2004; Matsuura and Pluckthun 2003; Schimmele et al. 2005; Zahnd et al. 2007), yeast display (Kondo and Ueda 2004) but the system most successful is phage display.
a suitable vehicle for displaying but also a biological template for nanotechnology, such as in the directed synthesis of semiconducting/magnetic nano-wires and lithium ion battery electrodes (Khalil et al. 2007; Nam et al. 2006).
The M13 wild-type virion is 65 Å in diameter and 9300 Å in length and contains an
ssDNA of about 6400 bp, coated with 2700 copies of the major coat protein pVIII
arranged in helical symmetry. The number of pVIII copies can change in a given range according to changing in DNA length. If phage DNA is elongated, as result of DNA insertion, pVIII copies increase to compensate the increased length of genome. Up to 12000 bases can be added to the wild-type phage genome without disrupting packaging.
Each end is capped with five copies of two minor coat proteins each one. At the proximal end of the virion (the first to cross the membrane when the phage leaves the host cell), five copies of pVII and of pIX form a 30 Å cap. These two small hydrophobic peptides play a role in the early stages of phage assembly, where they serve as a nucleus for the subsequent deposition of pVIII. In their absence, almost no phage particles are formed. The distal end of the virion terminates with five copies of pIII and of pVI forming cylindrical shape with a pointed end, and pIII extends further as a thin protrusion. pVI is the only coat protein exposing its carboxy terminus on the phage surface, the other protein are inversed oriented (Cabilly 1999; Kehoe and Kay 2005). These proteins are required for terminating the deposition of pVIII during phage assembly and for the host cell infection (for phage proteins functions, see table1).
Lifecycle
The M13 and other F pilus-specific filamentous phage life cycle begin with the infection of the host cell (Fig11) and precisely with the contact between F pilus and pIII protein.
The F pilus is an elongated helical array of protein subunits extending out from the cell wall. It is required for conjugal transfer of F plasmid DNA from a donor cell into a recipient bacterium (Harrington and Rogerson 1990).
Phage pIII protein structure consists of 3 domains, N1, N2 and C, connected by flexible linker regions (Fig.9). The carboxy terminus (C domain) anchors the entire protein to the phage, while amino termini, N1 and N2 domains, contact the F pilus.
Fig9. Domain organization of minor coat protein pIII, from (Kehoe and Kay 2005)
The binding of the N2 domain to the F pilus initiates the infection, disrupting interactions between N1 and N2 and their horseshoe shape (Lubkowski et al. 1998). After phage binding, the F pilus retracts until phage reaches the surface of bacterium and N1 binds to the E. coli membrane protein, TolA.
TolA A forms a complex together with TolQ and TolR on bacteria surface. The tolQRA complex spans the entire periplasmic space and is associated with adhesion between the inner and outer membranes.
Binding of the pIII domain N1 to the bacterial TolA causes phage disassembling and functional and/or conformational changes in the TolQRA complex, followed by separation of the outer and inner membranes, increasing of their permeability, and expansion of periplasmic space. The increased permeability might lead to the stable location of pVIII into the periplasm, as seen by biotin-marked pVIII experiments (Nakamura et al. 2003) and might be part of the driving force to move the phage
The (-) strand of this replicative form (RF) is the template for transcription, and the resulting mRNA molecules are translated into the 11 M13 proteins. One of these phage proteins, pll, nicks the (+) strand in the RF at a specific intergenic region. The resulting 3’ –hydroxyl acts as a primer for synthesis of a new viral strand via a “rolling-circle” mode of replication that uses bacterial enzymes. After one round, pll circularizes the viral (+) strand DNA, which then is converted to a covalently closed, super coiled, double-stranded progeny RF molecule by bacterial enzymes. In this way, a pool of progeny double-stranded RF molecules are produced that can direct the synthesis of the phage proteins.
The structural proteins pVIII, pVII, pIX, pVI, and pIII spontaneously insert into the inner bacterial membrane upon synthesis and await the replication of ssDNA genomes.
pIII is synthesized with a short amino-terminal signal peptide, which is removed after membrane insertion. The region spanning the membrane is part of the carboxy-terminal portion of the 150-residue CT domain, which is involved in anchoring pIII to the end of the phage particle.
Even pVIII has a signal peptide that directs its spontaneous insertions into the cytoplasmic membrane and that is removed after the insertion.
There are evidences (Nagler et al. 2007) that the helix-shape, membrane-spanning region of pVIII lays tilted in the inner membrane. The tilted helices could then interact with the neighbouring helices, resulting in close contacts and formation of sheet structures into the membrane.
Fig10. Model for the assembly of pVIII coat oligomers onto the nascent phage particle. The coat proteins form multimeric sheets in the inner membrane by protein-protein interactions and retain their tilt and their inter-protein contacts from the membrane to the viral particle. (Nagler et al. 2007)
When pV reaches sufficient concentrations, it forms dimers able to coat newly synthesized, single-stranded phage genomes, preventing their conversion to dsDNA.
pV-DNA structures contain approximately 800 pV dimmers and one single-strand viral DNA molecule(Webster 2004 ).
The DNA is wrapped inside the protein but is not completely enclosed by it. A small packaging signal, ssDNA that forms an imperfect hairpin, is left free of pV.
This hairpin is captured by a complex composed of the integral membrane proteins pIV, pXI, and pI. pIV forms a channel across the outer membrane, large enough to accommodate an extruding phage particle. Interestingly it does not let large molecules in or out of the periplasm in the absence of other phage proteins. pI and pIV are similar proteins both located in the inner membrane. It is assumed that they
The unique aspect of filamentous phage assembly is that it is a secretory process. Assembly occurs in the cytoplasmic membrane and nascent phages are secreted from the cell as they assemble.
Assembly begins when pV-DNA hairpin contacts pIV and the two minor coat proteins pVII and pIX are located at the tip of the phage particle.
During assembly, pV is stripped from the ssDNA in a process involving the
bacterium-encoded thioredoxin (TrxA), and the phage genome is coated with pVIII.
Since the tilted coat helix is found in the membrane-anchored protein, as well as in the phage particle (Nagler et al. 2007), probably only little structural rearrangements are necessary during the phage assembly process. Thus, it may simply occur by binding multimeric sheets of coat proteins out of the membrane (Fig10.).
Table1. F-specific filamentous phage genes/proteins and proprieties (Russel 2004)
The assembly continues until the end of the viral DNA has been coated by pVIII. At the end, it is incorporate in the nascent phage particle a complex formed by five copies of both pIII and pVI.
The release of phage requires a 93 residues C-terminal segment of pIII and it consists of conformational change in the pIII-pVI complex that detaches the complex, and the phage, from the cytoplasmic membrane.
It is important to note that M13 is a non-lytic bacteriophage, and the host cell is not killed by the infection. The first progeny phage particles appear in the culture supernatant about 10 min after infection (at 37°C). Their numbers increase exponentially for about 40 minutes, after which the rate becomes linear. About 1000 phage per cell are produced in the first hour. Under optimal conditions, the infected cells can continue to grow and divide- and produce phage- indefinitely. Persistent infection is possible because the five capsid proteins and the viral single strands are removed from the cell, by assembly and secretion, at a rate commensurate with their synthesis, and over-accumulation is avoided.
Phage display systems
Phage display systems can be grouped into two classes on basis of the vector system used for the production of phages.
True phage vectors are directly derived from the genome of filamentous phage (M13, f1, or fd) and encode all the proteins needed for the replication and assembly of the filamentous phage. In these vectors, the foreign DNA is ether cloned as a fusion with the coat protein present in the phage genome (Smith and Petrenko 1997) or inserted as fusion gene cassette with an additional copy of the coat protein. The resulting phage progeny presents respectively only the fusion coat protein or the wild type and the fusion coat proteins at the same time.
The second group of phage display systems utilizes phagemid vectors (Barbas et al. 1991; Hoogenboom et al. 1991); they contain a gene coding for one of the structural proteins to which foreign DNA can be linked allowing the translation of fusion proteins.
A phagemid is a special kind of plasmid that bears beside the plasmid origin of replication to replicate in E.coli hosts and the antibiotic resistance gene for the selection of plasmid-bearing cells, a phage-derived origin of replication, the intergenic region. This allows to replicate during phage life cycle and contains a hairpin section (packaging signal), which promotes the packaging of the ssDNA into the phage progeny (Russel and Model 1989). Once libraries are cloned into the plasmid, the phagemid DNA is used to transform bacteria where it can be amplified and from which it can be easily isolated, as well as any other plasmid. Production and maintenance of the library in bacteria, which replicate their DNA much more accurately than bacteriophage, is one way to preserve the diversity of the library.
Phagemid lacks almost all genes coding for phage proteins therefore it is not able to
produce progeny by itself. For the purpose of phage display, these proteins are
provided by super infecting phagemid-carrying cells with a helper phage. In this procedure, often called “phage rescue,” the helper phage provides all the proteins and enzymes required for phagemid replication, ssDNA production and packaging, and the structural proteins forming the phage coat also. The replication and packaging machinery supplied by the helper phage acts on the phagemid DNA and on the helper phage genome itself.
Therefore, two distinct types of phage particles with different genotypes are produced from cells bearing phagemid and helper phage DNA: those carrying the phagemid genome and those carrying the helper phage genome. Phage particles containing the helper phage genome are useless in phage display processes because they lack the genetic information on the exposed peptide. It is possible to modify the helper phage genome giving it a defective origin of replication or packaging signal, in this way there is a preferential packaging of the phagemid DNA over the helper phage genome (Kehoe and Kay 2005; Paschke 2006).
All five capsid proteins in the phage virion have been utilized for display purposes. Although peptide fusions to the amino or carboxy termini of other coat proteins as well as pVII, pIX and pVI have been reported (Gao et al. 2002; Hufton et al. 1999), the most common approach for peptide display is to fuse the foreign sequences to the amino terminus of pIII or pVIII.
These structural proteins all insert into the membrane prior to phage assembly, so the displayed peptide is completely exposed to the periplasmatic environment. This allows disulfide bond formation in both protein and peptide libraries.
Phagemid systems yield hybrid phage usually displaying wild type and fusion coat protein at a certain ratio. However, it was seen that during phage assembly wild type pIII (as well as the other wild type proteins) is preferentially incorporated, so that most phages exhibit the wild type phenotype.
That creates a problem in all selection systems, because phage particles can bind non-specifically to the solid support onto which the antigen is attached.
If non-displaying phages are propagated as well as displaying, selection results invalidated by false positive results. One solution to the problem is to avoid the delivery of wild type pIII by engineering the helper phage. Several helper phage constructs with a deleted or optionally untranslated gene III were developed (Baek et al. 2002; Duenas and Borrebaeck 1995; Kramer et al. 2003; Rakonjac et al. 1997;
also display multiple copies of the foreign proteins on phage surface, bringing to the possibility of cross-reactions between them.
Another strategy to reduce the possibility of false negative caused by non-displaying phages was reported by Kristensen and Winter (Goletz et al. 2002; Kristensen et al. 2000; Kristensen and Winter 1998). They introduced a protease cleavage sequence in the helper phage pIII protein between its N-terminal (N1– N2) domains and the CT domain. As seen, the N-terminal domain of pIII is required for infection, therefore phages with proteolytically cleaved off N1–N2 are noninfectious. This helper phage called KM13 does not alter the display rate, but it has not infectivity, thus only phages displaying the pIII fusion protein remain infectious, the others will be lost from the selection. Using these modified helper phages significantly reduced the background binding allowing selection of specific binders after one round of selection.
For all phage display systems, stringent control of fusion protein expression during all propagation steps appears to be crucial as background expression of pIII fusion proteins increases the metabolic burden or may even be toxic to the cells. Thus, leaky expression control generates a selection pressure leading to a higher frequency of recombination events and a growth advantage for non-functional clones, which in turn can lead to enrichment of these so-called “escape” mutants.
Most phagemid vectors rely on the lac promoter for expression of the pIII fusion, a promoter with a strong repressor when inactivated and inducible by adding lactate or IPTG (isopropyl β-D-thiogalactoside) a commonly used lactate derivate.
Antibody phage display systems.
Phage display was originally invented for the affinity selection of protein fragments expressed from cDNA fragments (Smith 1985); subsequently, phage display libraries for the affinity selection of peptides and antibody phage libraries were developed (Hoogenboom et al. 1991; McCafferty et al. 1990; Scott et al. 1990) (Breitling et al. 1991; Devlin et al. 1990; Hoogenboom et al. 1991; Marks et al. 1991; Scott et al. 1990) which are probably now the most widely used applications of phage display.
Antibodies were successfully selected using true phage systems (McCafferty et al. 1990; O'Connell et al. 2002) and pVIII fusion (Chappel et al. 1998; Kretzschmar et al. 1995) but the most useful antibody phage display systems are based on phagemid vectors encoding the antibody fragment library (as Fv, scFv or Fab) fused to the minor coat protein pIII.
Antibody fragments are preferred to the complete antibody because they are small and have high tissue penetrability, while maintaining their affinity and specificity. They are easier and faster to produce in recombinant form (Pansri et al. 2009) and it has been shown that both Fab(de Haard et al. 1999; Orum et al. 1993) and scFv (Clackson et al. 1991; Marks et al. 1991; McCafferty et al. 1990) can be expressed in the surface of M13 without apparent loss of the antibody's specificity and affinity. Antibodies are globular plasma proteins also known as immunoglobulins (Igs). Their basic functional unit is Ig monomer (Fig12 and 13) but they can also be organized in dimers, tetramers or pentamers depending of their biological properties, functional locations and ability to deal with different antigens. These different structural organizations are called antibody isotypes.
The monomer is a Y shaped molecule consisting of four polypeptide chains: two identical heavy chains (H) and two identical light chains (L) held together by disulfide bridges and noncovalent bonds.
Each heavy chain has two regions, the variable region (VH) and the constant region (CH), formed by three globular domains, CH1, CH2, and CH3. The constant region is identical in all antibodies of the same isotype, but differs in different isotypes; indeed mammalians have five types of heavy chain, specific for each isotype, differing in size and composition.
The variable region of the heavy chain differs in antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or B cell clone.
Fig.12 Schematic diagram of a typical antibody showing two Ig heavy chains (blue) linked by disulfide bonds to two Ig light chains (green). The constant (C) and variable (V) domains are shown.
The C regions of both heavy and light chains form the antibody structural domain, while the other regions form the functional domain: pairing of the heavy and light chain variable regions creates an antigen-binding site (paratope) which recognizes a single antigenic determinant (epitope).
Discrete Antibody domains can be separated by protease digestion or produced by recombinant technology; this allows the production of antigen-binding fragments, named Fab and Fv, to use in phage display systems (Fig14 and15). The larger Fab (fragment antibody) consists of VH-CH and VL-CL segments linked by disulfide bonds. The smaller Fv (fragment variable) is composed of the VL and VH regions only. The recombinant version of the Fv is termed the single-chain variable fragment (scFv). The two variable regions in the scFv are artificially joined with a flexible peptide linker, usually a 15 aa Gly-Ser, and expressed as a single polypeptide chain. The linker allows the association of the VH and VL to form the antigen-binding site.
Fig.13 The antibody structure. The heavy chains are in red and blue, the light ones are in yellow and green.
Fig.14 The scFv structure. In blue the variable domain of the heavy chain, VH and in yellow thevariable domain of the
Fig.15 Antibody fragments. Representation of normal human antibodies and fragments The variable region is indicated by the grey colour, while the black represents constant regions.
Virtually all epitopes can trigger an antibody response. Successful recognition and eradication of many different type molecules requires diversity of aminoacids composition among antibodies (Mian et al. 1991).
Although a huge repertoire of different antibodies is generated in a single individual, the number of genes available to make these proteins is limited. Several complex genetic mechanisms have evolved that allow vertebrate B cells to generate a diverse pool of antibodies from a relatively small number of antibody genes (Nemazee 2006), one of these mechanism is a somatic recombination, called V(D)J recombination from the name of the genetic site.
The variable region of each immunoglobulin heavy chain is encoded by several gene segments: variable (V), diversity (D) and joining (J) segments (Nemazee 2006). Even the light chains have the same gene partition but they lack the D segments, having only V and J ones. As showed in Fig.16 multiple copies of the V, D and J gene segments exist.
Fig16 Simple overview of V(D)J recombination of immunoglobulin heavy chains; for the light chains the process is the same but there is not D segments.
In the bone marrow, in each developing B cell, precursor of Abs producing-cells, the first recombination event to occur is between one D and one J gene segment of the heavy chain locus. Any DNA between these two genes is deleted. This D-J
of the heavy chain and both the constant mu and delta chains (Cμ and Cδ). Following processes will involve a constant chain choice, called antibody class selection and depending on the type of Abs-producing cell.
The kappa (κ) and lambda (λ) chains of the immunoglobulin light chain loci rearrange in a very similar way: the first step of recombination for the light chains involves the joining of the V and J chains to give a VJ complex before the addition of the constant chain gene during primary transcription. Translation of the spliced mRNA for either the kappa or lambda chains results in formation of the Ig κ or Ig λ light chain protein.
After a B cell produces a functional immunoglobulin gene during V(D)J recombination, it cannot express any other variable region (a process known as allelic exclusion) thus each B cell can produce antibodies containing only one kind of variable chain (Bergman and Cedar 2004; Janeway 2001).
V(D)J recombination process is carried out by a collection of enzymes some of which are expressed in many cell types , some other are lymphocyte specific, as the recombination activating gene-1 and -2 (RAG-1 and RAG-2) that start the recombination process.
Other mechanisms responsible for antibody diversity include:
• a junctional diversity due to the imprecise joining mechanisms and to deletion or addition of random nucleotides during the V(D)J recombination;
• the possibility of an enlargement of the architecture of the paratope by adjusting the angle between the associated VH and VL domains;
• the specific maturation of the paratope caused by a somatic hypermutation that improves the shape complementarity of the antibody with the antigen. The cumulative effects of these mechanisms determine the antigenic affinity and specificity of an antibody. The construction of recombinant antibody uses some of the same mechanisms to the production of Abs phage libraries.
Construction of scFv phage libraries
The construction of recombinant Antibody libraries is based on the mimicry of the process used by immunosystem to select and enlarge their Abs repertory.
In mammals the Pre B cell commits forming a B cell and antibody gene recombination occurs so that naive B cells with low affinity antibody receptors are produced. Then, through interaction with the antigen, B cell antibody undergoes affinity maturation and isotype switching, giving rise to soluble antibodies with the highest affinity for each antigen.
Following this scheme, protocols for antibody libraries construction have been optimized; three main types of antibody libraries exist, depending on the source of genetic material and way of construction, these are immune, naive and semi-synthetic libraries (Winter et al. 1994).
In immunized library, V-genes are derived from the immunoglobulin mRNA of B-cells from an immunized animal or, in certain cases, human. Humanized immune repertoires can also be prepared using immunized transgenic mice (xenomice). The antibody genes can be recovered from the B-cells by PCR, amplified and then cloned into phages or phagemids.
An immune antibody library has two main characteristics: it will be enriched in antigen-specific antibodies, and some of these antibodies will have undergone affinity maturation by the immune system (Clackson et al. 1991).
High-affinity antibodies were reportedly derived from mice (Andersen et al. 1996), chickens (Yamanaka et al. 1996), and rabbits (Lang et al. 1996). Antibody libraries were also derived from sheep (Charlton et al. 2001), cows and nonhuman primates (Tordsson et al. 2000).
Disadvantages of immune libraries include the long time required for animal immunization, the lack of immune response to self or toxic antigens, the unpredictability of the immune response to the antigen of
A unique application of immune libraries is to clone high-affinity antibodies present after viral infections or cancer, and antibodies to self-antigens present in patients with autoimmune diseases. Analysis of such antibodies could aid in the identification of antigenic epitopes involved in the humoral immune response.
Using oligonucleotide directed mutagenesis or PCR-based techniques synthetic repertoires can be made by randomizing the variable region of the heavy chain, the most diverse in composition and length region and the most central to the antigen-binding site.
A major advantage of synthetic repertoires over naı¨ve ones is the potential to control and define the contents, local variability and overall diversity of synthetic libraries (Akamatsu et al. 1993; Garrard and Henner 1993; Sanz 1991).
Naïve libraries has been constructed from the light-chain and heavy-chain IgM-V-gene pools of B cells isolated from peripheral blood lymphocytes (PBL), bone marrow, or spleen cells of non-immunized healthy donors (Pansri et al. 2009; Schofield et al. 2007). The mRNA of B-cells, encoding for rearranged immunoglobulins, it is amplified by PCR and cloned in phagemid vectors. It gives a representation of the repertoire of variable regions derived from all conceivable framework assemblies (Fig17).
The affinity of these antibodies was similar to that seen in naı¨ve primary immune response and was sufficiently reactive in Western blot, ELISA (enzyme-linked immunoabsorbent assay), and FACS (Fluorescence-activated cell sorting) analysis (Nissim et al. 1994).
Key advantages of these libraries include the possibility to isolate human antibodies to self, non-immunogenic or toxic antigens. They are useful because of a single library can be used for all antigens and because of the few time needed for antibody generation (2—4 rounds of selection in two weeks). Furthermore, they give the possibility to direct isolate high affinity antibodies when very large repertoires are used without any immunization or affinity maturation required.
Disadvantage of naı¨ve libraries could be an unequal expression of the V-genes repertoire, an unknown history of the B-cell donor, and a potential limited diversity of the IgM repertoire which can influence the content and quality of the library. Moreover, a low affinity of antibodies isolated due to small sized libraries. Many approaches have been proposed to improve phage displayed human antibody repertoires, mainly by increasing the library sizes (Vaughan et al. 1996) by sophisticated in vivo (Cre-lox) recombination method (Griffiths et al. 1994), and by improving cloning steps (Sheets et al. 1998).
How Carry Out Selections
Antibody libraries are screened and enriched for antigen-specific clones by a technique known as bio-panning (Fig18) in which phages displaying scFv are incubated with an immobilized antigen of interest (Clackson et al. 1991; Nissim et al. 1994).
The ligand used to screen the phage library has to be tagged or immobilized in a way that allows it to retain its biological function. In general the known or expected characteristics of the ligand will dictate the immobilization procedure used, which usually is the direct or peptide-mediated binding to an affinity column, or to the plastic surfaces of immunotubes or enzyme-linked immunoadsorbent assay (ELISA) plates. However some ligands were used in solution (Hawkins et al. 1992), exposed on cell surface (Kupsch et al. 1999; Mintz et al. 2003) or directly injected into animals for selection in their own tissue (Kolonin et al. 2002; Pasqualini and Ruoslahti 1996; Yao et al. 2006; Zurita et al. 2004).
Using immobilized targets, the phage library is incubate into the immunotube or ELISA plate and then washed away. Thus unbound phages are removed by washing whereas phages displaying scFv that specifically bind the antigen are retained by their binding.
Specific phage-displayed antibodies can be eluted from their antigens with acidic solutions (such as HCl or glycine buffer), with basic solutions such as tri-ethylamine, by enzymatic cleavage of a protease site constructed between the antibody and pIII (Rhyner et al. 2004; Ward et al. 1996), or by competition with excess antigen.
To prepare phages for a further round of affinity enrichment, eluted phages are simply used to infect the E.coli cells. Several round of affinity enrichment are required to select specific phagemids from background binding. Enrichment is dependent on many factors including the quality of the library, the affinity of the ligand for target and the number of specific ligands available for a specific target. To produce soluble scFv, antigen-positive phages are used to infect a specific strain of E. coli (e.g., E. coli HB2151) that will direct production of soluble scFv. Such E. coli strain is termed “nonsuppressor” cells as they recognize an amber stop codon, engineered between the scFv gene and pIII gene (g3) in the phagemid, and only
express the scFv without the pIII protein. The phagemid is also designed to introduce polyhistidine tag fused to the expressed scFv, thereby permitting rapid and simple protein purification.
Depending on the isolated clone, soluble antibodies may be present in the culture supernatant, the bacterial periplasm, and/or inside the bacterial cells. All three fractions must be isolated and analyzed in a Western blot, using a commercially available conjugated anti-histidine (His) tag antibody, to determine the location of the soluble antibodies. Soluble scFv are relatively simple to isolate, can be economically produced in bacteria in very large quantities (Mavrangelos et al. 2001; Yu et al. 2005), and do not entail complex refolding procedures (Azzazy and Highsmith 2002; Sanchez et al. 1999).
Materials & Methods
Antigen productionPCR amplification and cloning
Primers were designed with the required flanking sequences using the Vector NTI program. The obtained primers were about 30 nucleotides (nt) length inside the coding sequence of gene, giving a total length of about 45 nt for each one.
Sense primer flanking sequence:
5' GAC GAC GAC AAG ATX* – insert-specific sequence - 3' Antisense primer flanking sequence:
5' GA GGA GAA GCC CGG TXX**– insert-specific sequence - 3'
The cDNAs used for the PCR amplification and antigen production came from the German Resource Center for Genome Research RZPD (Deutsches Ressourcenzentrum für Genomforschung). They sent the full length proteasome subunit specific cDNA sequences cloned into bacterial vectors that were amplified in liquid cultures, purified and store in the freezer. Then purified plasmids were used for the PCR amplification.
PCR reaction was performed adding in a PCR tube:
• 5µl of 10x PCR Buffer, for a final concentration of 1x
• 1µl of Deoxynucleotide Mix, for a final concentration of 200 µM
• 2µl of Forward Primer, for a final concentration of about 12.5 pM
• 2µl of Reverse Primer, for a final concentration of about 12.5 pM
• 1µl of Template cDNA
• 0.5µl of Taq DNA Polymerase for the final concentration of 0.05 units/µl
Tubes were run at the settled program, but with different annealing temperatures because of the different chemistry of primers and their own Tm (temperature of melting).
PCR cycle:
Heated Lid 110°C
Preheat Lid Off
Pause Off
Denaturation 94°C for 5 minutes
Hot Start Off
Loop
30 Cycles Denaturation 94°C for 30 seconds Annealing 50-60°C for 30 seconds Elongation 72°C for 2 minutes
Final Extension 72°C for 5 minutes
Final Hold 10°C
PCR was verified by electrophoresis on agarose gel: in a 1% agarose gel, 1µl of Loading Buffer and 4µl of sample (PCR products) were loaded for each well; in the 6th well 5 µl of DNA marker was loaded. The electrophoresis was run at 500 volts for one hour.
PCR products were purified by Wizard SV Gel and PCR Clean-Up System DNA purification kit. This kit has a membrane that can selectively bind DNA and allow to wash and to clean the sample. After washes, samples were centrifuged to separate DNA from impurities, and eluted with Nuclease-Free water.
The samples were then diluted 1:100 and used on the spectrophotometer to evaluate their concentration. Measures were done at 260 nm and 280 nm and concentration calculated according to the equation 1 OD260 = 50 ng/μl for dsDNA
Then the ratio OD260/ OD280 was used to get an indication of purity of the sample. All samples give a purity upper than 1.5.
at 72°C makes the enzyme inactive. Annealing required 5 minutes incubation with the plasmid.
Transformation takes place by heat-shock, adding 1μl of plasmid solution to 20μl of just de-frozen competent cells and putting the mixture at 42°C. After 30 seconds samples can put back on ice, add to SOD medium and incubate at 37° on shaking for one hour, so bacteria can recover before plating on TYE + ampicillin selective medium. The table2 summarizes the protocol.
Table2. Annealing and insertion protocol.
Actions Substances Quantities
Mix 0.02 pmol/ml sample 5μl
10x T4 DNA polymerase
Buffer 1μl
25 mM dATP solution 1μl
100 mM DDT 0.5μl
2.5 U/μl T4 DNA polymerase 0.2μl Nuclease free water final volumeFor 10μl of Incubate at RT for 30 minutes
Incubate at 72° for 20 minutes
Add TriEx-4 Ek/LIC 1μl
Incubate at RT for 5 minutes
Add and mix very well 25mM EDTA 1μl
Put on ice
Mix Plasmid solution 1μl
De-frozen competent cells 20 μl Incubate on ice for 5 minutes
Heat in water bath at 42° for 30’’ and back in ice
Add SOD medium 80μl
Incubate at 37° for 60 minutes
Plate on TYE+Amplicillin
plates 50μl for each plate
Incubate at 37° overnight
Four clones for each sample were amplified by PCR from the transformed bacteria plates and the insertion was verified by electrophoresis using a DNA 100bp marker. Samples resulted on the expected base length. A glycerol stock of each positive clone was done.
The better clones were chosen for plasmid purification by QIA prep Spin Kit. The kit lyses bacteria under alkaline conditions and then neutralizes the lysate with a high-salt binding solution. After lysate cleaning, a silica selective membrane absorbed the plasmid, allowing wash away everything else. Samples are then eluted from the membrane using a low-salt solution.
To test this purification an electrophoresis gel was run, after one hour incubation at 37 °C with XbaI restriction enzyme to linearize the plasmids.
After the purification, an aliquot (10μl of each sample) of plasmid solution from each clone was sequenced by the MWG Company and the results were compared to ones published on Gene Bank by CLC Combined Work Bench software.
Proteasome subunits expression
For antigens expression Origami B(DE3)pLacI cells were used. The transformation was made by heat-shock adding 1μl of plasmid solution to 20μl of just de-frozen Origami B(DE3)pLacI cells and putting the mixture at 42°C for 30 seconds before to be incubate at 37° on shaking for one hour, and then plated on TYE medium containing:
• 15 μl/ml of kanamycin,
• 12.5 μl/ml of tetracycline,
• 34μl/ml of chloramphenicol and
• 1 μl/ml of ampicillin, to select bacteria harbouring the pTriEx-4 Ek/LIC plasmid.
Because of antibiotics influence, cells grew slowly. The transformation was tested by PCR amplification of a colony picked up from plates and primers specific for the genes inserted in the plasmid.
