Ubiquitin is a small highly conserved regulatory protein that is universally present in all organisms (Goldstein et al., 1975). It is composed of 76 amino acids with a single 8.5 kDa polypeptide chain (Vijay-Kumar et al., 1985) and has been reported not to be found in prokaryotes. It is highly conserved meaning that there is no much difference in the amino acid sequence when compared in different organisms. Ubiquitin is particularly of great interest in research because of its unparalleled sequence conservation and its resistance to tryptic digestion. Also, studies have shown that it is quite stable over a wide range of pH and temperature values. It is found throughout the cells hence the name 'ubiquitous'. Ubiquitin can exist either freely or with other proteins as part of a complex. It is usually conjugated to proteins through a covalent bond between the glycine at its C-terminal end and the side chains of lysine on the proteins in a process that depends on ATP hydrolysis.
Ub's functions are
1.1. Structure of Ubiquitin
Ub is a heat-stable protein that folds up into an extremely compact monomeric globular structure. It is tightly bonded by hydrogen, 87% of the polypeptide chain are approximately involved in hydrogen-bonded secondary structure. In 1987, Vijay-Kumar et al., determined the structure of ubiquitin refined at 1.8?? resolution. Its features include three and a half turns of ??-helix, a short piece of 310-helix, a mixed ??-sheet that contains five strands, and seven reverse turns. There is a marked hydrophobic core formed between the ??-sheet and ??-helix (Vijay-Kumar et al., 1987). The tyrosine, histidine, and two phenylalanine residues are situated on the surface of the ubiquitin molecule (Vijay-Kumar et al., 1985). Ubiquitin possesses a total of 7 lysine residues, in polyubiquitination, lysine 48 is the normal point of attachment for another ubiquitin molecule.
1.2. Ubiquitination pathway
Although proteins are required for structural and biochemical requirements of the cell, they are also broken down in a regulated process. Proteins can be long-lived or short-lived depending on the nature of the amino acids present at their N-termini. Proteolysis enables the cell to get rid of misfolded or damaged proteins and also to adjust the concentration of essential proteins in the cell. This degradation can be accomplished through the attachment of one or more Ub molecules to a target protein in a process known as Ubiquitination. The conjugation of Ub onto a protein can be termed as the 'kiss of death' because it tags the labelled protein for degradation in the proteasome, a barrel-shaped protein complex where proteases act on the protein.
Ubiquitination is a process of targeting protein for different biological processes such as proteosomal degradation, endocytosis, virus budding, and vacuolar protein sorting (Vps). Ubiquitinated proteins are recognized using conserved ubiquitin binding modules. One (monoubiquitination) or more (polyubiquitination) ubiquitin molecules can be attached to a target protein. However, ubiquitin cannot just be conjugated to an abnormal protein even in the presence of ATP, this is where the 3 enzymes come into play. Ubiquitination is an enzymatic, protein post-translational modification process that involves the sequential catalytic action of three ubiquitin enzymes E1, E2 and E3. The reaction entails the transfer of a covalent bond with ubiquitin from one enzyme to another and finally to a target protein. E1 enzymes are known as Ub-activating enzymes, they change Ub to a reactive state to increase the chances that its C-terminal glycine will react with the lysine side chain on the target protein. The transfer of Ub to its active site, the amino acid cysteine requires ATP and is thus an energy-dependent process. Ub is then passed on to E2 enzymes known as Ub-conjugating enzymes to catalyse the binding of Ub to the target protein. E3 enzymes known as Ub-ligases collaborate with E2 enzymes and help to recognize the target protein. The process can be repeated until a short chain with three or more Ub molecules is formed to target the protein to the proteasome.
Ubiquitination is a reversible process, and the cleavage of ubiquitin from substrates is performed by specific deubiquitinating enzymes called DUBs (Hochstrasser, 1995) or deubiquitinases.
Several substrates have been identified for the ubiquitination pathway. Examples are cyclins which are involved in the control of the cell cycle, p53 ' the tumour suppressor protein and NF-kB ' the transcription factor associated with inflammation and immune response. There is a corresponding E3 ligase for each substrate.
1.3. Functions of Ubiquitin
Ubiquitin (originally known as ubiquitous immunopoietic polypeptide) was first identified in 1975 as a protein of unknown function expressed universally in living cells. The main function of ubiquitin is to regulate the degradation of specific proteins; it serves as a tag that marks protein for degradation by the proteasome. Proteins that are to be degraded are first conjugated with Ub and then transported to the proteasome for degradation. Ub-linked regulation is energetically costly because the protein has to be re-synthesized if it is needed again. The ATP-dependency is because of the need to specifically target the proteins needed for degradation by the 26S proteasome which is an organelle in the cell for degrading and recycling unneeded proteins. In order for the condemned protein to be recognized by 26S proteasome, at least four Ub molecules must be attached to its lysine residues. The proteasome is a complex, barrel-shaped structure with two chambers, within which proteolysis occurs. Ub molecules are cleaved off the protein immediately prior to its destruction and are recycled for further use.
It was observed that variations in Ub attachment could control protein function without proteolysis. Ub labelling is not always disastrous for the protein, there are several non-proteolytic functions related with the attachment of ubiquitin. Ubiquitin tags also direct proteins to other locations in the cell, where they control other protein and cell mechanisms. Ub conjugation plays an important role in several cellular functions such as DNA repair, heat shock, chromatin structure, embryogenesis, transcription regulation, and apoptosis. Ubiquitin antibodies are used to identify inclusion bodies which are abnormal accumulations of protein markers of diseases inside the cells. Examples of such abnormal inclusions in cells are seen in diseases such as Alzheimer and Parkinson
Table 1: Chemicals used
Overexpression of the protein
LB (Lysogeny Broth) - media 10 g of Casein Hydrolysate; 5 g of Yeast extract; 10 g of NaCl; were dissolved in 1 L of distilled water. For agar plates, 20 g of agar were added to the mixture prior to dissolving. The solution was autoclaved at 125??C and let to cool after which 1 mL of ampicillin(100 mg mL-1) was added.
M9 - minimal media 6 g of Na2HPO4'2H2O; 3 g of KH2PO4; 1.5 g of (15NH4)2SO4; 2 g of 13C- Glucose; 0.5 g of NaCl; 1 mL MgSO4 (1 M); 10 mL trace element solution; 1 mL vitamin mixture (100x); 100 ??L CaCl2 (1 M) were dissolved in 1 L of distilled water. The solution was then sterilised using a millipore filter (0.22 ??m). 1 mL of Ampicillin (100 mg mL-1) was then added before its use.
Trace Element Solution
5 g EDTA; 0.83 g FeCl3; 0.084 g ZnCl2; 0.013 g CuCl2'2H2O; 0.01 g CoCl2'6H2O; 0.01 g H3BO3; 0.0002 g MnCl2'4H2O were dissolved in 100 mL distilled water after which the solution was autoclaved.
Vitamin Mixture (100x) 100x BME Vitamins solution from Sigma Aldrich stored at -20??C.
Ampicillin stock solution
A 100 mg mL-1 Ampicillin solution was prepared using 18 M?? water. 1 ??L of ampicillin was used per 1 mL of the culture media. It was stored at -20??C
1 M IPTG-solution (Isopropy l - ?? - D - 1 - thiogalactopyranoside) A 238 mg mL-1 IPTG solution was prepared using 18 M?? water. To induce the protein expression, 1 ??L of IPTG solution was used per 1 mL of the culture media. The solution was stored at -20??C
100 mM AEBSF Protease inhibitors 240 mg of AEBSF was dissolved in 10 mL of 18 M?? water. It was stored at -20??C.
75 g of glycine; 15 g of Tris and 5 g of SDS were dissolved in 1 L of distilled water to make a 5x concentrated buffer. Before use, it was diluted to 1x.
Ammonium Persulfate (APS) A 10% APS solution was prepared by dissolving 1 g of ammonium persulfate in 10 mL 18 M?? water. Storage was at 4??C.
5x Loading Buffer 0.6 mL of 1 M Tris @ pH 6.8; 5 mL of 50% Glycerol; 2 mL of 10% SDS; 1 mL of 1% Bromophenol blue; 0.5 mL of Dithiothreitol (DTT); 0.9 mL of distilled water were mixed and stored at 4??C.
Resolving Gel Buffer
75 mL of 2 M Tris @ pH 8.9; 4 mL of 10% SDS and 21 mL of distilled water were mixed and stored at 4??C.
Stacking Gel Buffer 50 mL of 1 M Tris @ pH 6.8; 4 mL of 10% SDS and 46 mL of distilled water were mixed and stored at 4??C.
A 30% solution of acrylamide/bisacrylamide mix stored at 4??C.
Staining Solution 1 g of Coomassie Brilliant blue was dissolved in 450 mL of methanol followed by the addition of 450 mL of distilled water and 100 mL of concentrated acetic acid.
Destaining solution Mixture of 800 mL of distilled water, 100 mL concentrated acetic acid and 100 mL of Methanol.
Binding Buffer (50 mM Na-Acetate; 5 mM EDTA @ pH 4.5) 6.80 g of Na-Acetate and 1.86 g of EDTA (Ethylenediaminetetraacetic acid) were dissolved in
1 L of distilled water, the pH was adjusted to 4.5 and the solution was sterilized using a Millipore filter. 20 ??L of 2.5 M NaN3 solution was then added in order to prevent microbial growth. Storage was at 4??C
Elution Buffer (50 mM Na-Acetate; 5 mM EDTA; 0.6 M NaCl @ pH 5.0)
6.80 g of Na-Acetate; 1.86g of EDTA and 35.06 g of NaCl were dissolved in 1 L of distilled water, the pH was adjusted and the solution was sterilized using a Millipore filter. 20 ??L of 2.5 M NaN3 solution was added. Storage was at 4??C
NaN3 - solution A 2.5 M NaN3 solution was prepared in 18 M?? water by dissolving 1.6 g of NaN3 in 10 mL of 18 M?? water. Storage was at 4??C.
2.1. Cultivation of Ubiquitin in E.coli
The protein ubiquitin was first expressed in Escherichia coli. The isotopic labels 15N and 13C were introduced by growing the bacteria in M9 minimal media supplemented with 13C- Glucose and (15NH4)2SO4.With the help of IPTG (Isopropyl-??-D-1-thiogalactopyranoside), the expression was triggered and the harvested cells were lysed by ultrasonication to release the protein for purification.
2.1.1. Preparation of bacterial culture
In order to cultivate the protein, first the bacteria culture has to be prepared. LB-agar plates containing ampicillin were inoculated with 104 and 106 times diluted bacterial glycerol stock and grown overnight in an incubator at 37??C for 18 hours. The bacteria ('don't know the strain'?) is carrying a plasmid which is serving as a vector that will be used to introduce the protein gene that the bacteria will produce. It also has a gene resistant to antibiotics, this is useful because ampicillin will help to select the bacteria strains that have truly incorporated the plasmid as they will be resistant to the antibiotics and will be able to survive.
100 ml of already prepared ampicillin-containing LB media was divided into 3 smaller flasks. Each of the flasks was inoculated separately by a colony of the resistant growing bacteria from the LB-agar plates. They were then incubated overnight in a shaking incubator at 37??C at 200 rpm. The minimal media was autoclaved and cooled ('?) a flask containing 250 ml of the M9 media was inoculated with 3 ml of the starting culture and incubated in a shaker. While incubating, its optical density at 600 nm (OD600) was monitored periodically (every 30 minutes) using a pure minimal media as a blank. The optical density indicates the growth and aggregation of the cells.
2.1.2. IPTG Induction, harvesting and lysing of cells
In order to maximise the protein yield, the cells were induced for ubiquitin expression with IPTG during the mid-log phase of growth shortly after the minimal media reached an OD600 of 0.6. The induction period was for 24 hours at 28??C. Isopropyl ??-D-1-thiogalactopyranoside (IPTG) induces lac operon and recombinant gene expression in E. coli. The lac operon is used to create recombinant proteins in E. coli that can then be purified and studied in NMR. The gene of interest is usually inserted into a plasmid vector that contains a gene coding for antibiotic resistance and a lacI gene from the lac operon that codes for lac repressor (LacI). The gene of interest is inserted after the T7 promoter DNA sequence, the lac operator DNA sequence, and the ribosome binding site. The plasmid vector is short and circular and it is taken up by E. coli without replacing its existing genome. Therefore during cell divison, new copies of the host chromosome as well as new copies of the smaller cloning vector containing the gene of interest are made for every daughter cell.
After induction, the dense liquid media was centrifuged at 4000 g at 4??C and the cell pellets were collected on ice to avoid the degradation of ubiquitin. The pellets were re-suspended in a protease inhibitor containing binding buffer. Addition of protease inhibitor to the suspension helps to prevent the proteolytic degradation of the protein. To lyse the cells, the bacterial suspension was ultasonicated on ice. For best result, the sonication was carried out 5 times for 30 seconds burst each. The lysates were then collected and centrifuged for 45 minutes at 4000 g. To determine how successful the cell lysis process had gone, SDS-PAGE was employed to examine both the supernatant and the pellet.
SDS-PAGE is an electrophoretic technique that separates protein according to their size. In principle, it is based on the migration of charged protein molecules in an electric field which acts as a driving force to draw the SDS coated proteins towards an electrode of opposite charge. The different molecules will be separated depending on their relative mobility with larger proteins moving more slowly than smaller ones. Due to its synthetic, thermostable and chemically inert properties, polyacrylamide is the most widely used material for the separation of proteins. Under native condition, proteins are separated based on their charge, size and shape. However, the introduction of SDS an anionic detergent minimizes the charge contribution by producing even charges across the length of a linearized protein. It uniformly distributes charge over the protein molecule by binding to protein in a ratio of 1.4 g SDS per 1 g of proteins. In the end, the proteins have negative charges which mean that they will migrate to the anode in an electric field and can therefore be separated based on their size only. Also SDS solubilizes and linearizes protein by denaturing secondary and tertiary structures of protein to primary structure with the exemption of disulphide bridges. The disulphide bridges are reduced with the help of reducing agents like Dithiothreitol (DTT) or 2-mercaptoethanol. Other chemicals used in the preparation of the polyacrylamide gel are Ammonium persulfate (APS) and N, N, N', N'-tetramethylethylenediamine (TEMED). APS initiates the polymerization of acrylamide momomers and spontaneously decomposes releasing free radicals. TEMED helps to stabilize the free radicals as well as to promote polymerization.
PAGE is a polymer of acrylamide and bisacrylamide monomers, it is made up of an irregular network of tunnels through a meshwork of fibers. Acrylamide forms linear polymers while bisacrylamide forms crosslinks between polyacrylamide chains. Its preparation involves the casting of two different layers of acrylamide between glass plates. The separating or resolving gel which is usually the lower layer is of higher concentration and is responsible for separating the protein by size. The stacking gel which is the upper layer is usually of a lower acrylamide concentration and contains the wells into which the protein is loaded. The protein micelles are stacked in order to decrease the spread of the sample before the micelles enter into the separating gel. The proteins are then compressed into micrometer thin layers when they reach the separating gel. For effective separation, the right acrylamide concentration to use is usually determined based on the size of the protein of interest. For large proteins, 10% acrylamide is sufficient; however, ubiquitin is a small protein so the acrylamide concentration used for the experiment was 15%.
Immediately after pouring the gel mix into the glass plates, it is overlaid with ethanol. This will produce a levelled surface at the top of the separating gel ensuring that the bands are straight and uniform. After polymerization, the ethanol is poured off and the stacking gel is poured on top of the separating gel. The combs are gently inserted while avoiding bubbles under the teeth. After polymerization, the gel apparatus is placed in an electrophoretic tank and filled with 1x running buffer. The combs are removed and the wells are then washed with the running buffer. The sample was then loaded with the appropriate amount of 5x loading dye to make a volume of 15 ??l. To identify the proteins by their size, 3 ??l of a protein standard of known protein sizes is loaded with the samples and run along with it under the same conditions at 200 V for 40 minutes. For the visualization of the separated protein bands, dyes such as coomassie brilliant blue which binds non-specifically to the protein can be used to stain the gel for about 30 minutes. The gel is afterwards destained in a destaining solution for about 45 minutes
2.3. Purification of the protein
After the success of the cell lysis had been confirmed by SDS, the extract was purified using fast purification liquid chromatography (FPLC) with a sepharose column. The collected fraction of interest were then concentrated using Amicon Ultra filtration flasks form Milipore and further purified by size exclusion chromatography. The pure protein was obtained using ion exchange chromatography and size exclusion chromatography. During each purification step, the purity of the protein fractions from the chromatographic techniques was evaluated using SDS-PAGE. Finally, the concentration of the purified ubiquitin was determined using UV-vis absorbance spectroscopy and its folding was verified by protein NMR.
2.3.1. Ion Exchange Chromatography
Ion exchange chromatography is a charge-charge interaction between the proteins and the opposite charges in the immobilized groups in the IEC medium. It separates proteins according to the differences in their net surface charge. In cation exchange, cations bind to anions in the stationary phase while in anion exchange, anions will be attracted to the cations in the stationary phase. Most ion-exchange experiments are carried out in four different stages ' equilibration, sample application and wash, elution and regeneration. The stationary phase is first equilibrated to the desired start condition. Next the sample is loaded in order to bind the target molecules then the column is washed out to remove all non-binding protein i.e. the UV signal returns to the baseline. Uncharged proteins and proteins that are similarly charged with the ionic group are eluted during or just after sample loading. In order to bind all appropriately charged proteins, the sample buffer should have the same pH and ionic strength as the starting buffer. In the elution step, the buffer composition is altered in order to elute the bound proteins from the ion exchanger. A typical way is to increase the ionic strength of the buffer with NaCl or KCl or to change the pH in order to desorb the bound proteins. As the ionic strength increases, the salt ions (Na+ or Cl-) compete with the bound protein for charges on the surface of the medium leading to the elution of one or more of the bound species. The protein with the lowest net charge at the selected pH will be the first to be eluted from the column while the protein with the highest charge at a certain pH will be strongly retained and be eluted last. The higher the net charge of the protein, the higher the ionic strength required for elution. The regeneration step removes all molecules still bound to ensure that the full capacity of the stationary phase is available for the next round.
Ubiquitin is purified from the protein extract by employing IEC using a FPLC system with a SP sepharose column. To prevent the clogging of the column, the extract was first filtered to get rid of cell impurities using a GVS filter (1.2 ??m). The column was then equilibrated using the binding buffer at 2 ml/min. 10 ml of the protein extract were then injected into the colum at a flow rate of 1 ml/min. The whole set up was carried out in a fridge at 4??C. Shortly after the flow through was out of the column, the elution buffer was applied till the final concentration of 70%. The fractions were collected in 2 ml volume till no signal changes were observe in the chart plotter. The fractions were then examined on a 15% gel to determine the fraction that contains ubiquitin. Those containing ubiquitin were collected together and the volume was reduced to about 6 ml by centrifuging the collected fractions in an Amicon Ultra filtration flask (pore size 3 kDa) at 4000 g at 4??C
2.3.2. Size Exclusion Chromatography
SEC separates analytes based on their size. Unlike other liquid chromatography mechanisms like IEC, a unique feature of SEC is that there are no actual interactions of the analytes with the stationary phase. Instead it separates the analytes based on molecular sizes. The sample is injected into a column packed with porous particles of fairly defined pore size. The mobile phase is usually the same buffer as that used for the sample. Separation is attained by the differential exclusion of the sample molecules from the pores of the packing surfaces as they travel through a bed of porous particles. This depends on the ratio of molecular dimensions to the distribution of pore-size diameters. Large molecules which are not able to penetrate through the pores of the packing surfaces are eluted through the void volume of the column. Small molecules can freely diffuse in and out of the pores therefore their elution take longer. Molecules with high molecular weight elute first followed by low molecular weight molecules.
The chromatographic set up used for IEC was also used for SEC at 4??C. However, the column was packed with porous HighLoadSuperdex 75 particles which are dextran-cross-linked agarose. The concentrated protein solution was further purified by loading it into the column ensuring that there are no bubbles and the flow rate was set to 0.5 ml/min. Shortly after a peak was detected on the chart plotter, the fraction was set to 2 mL. Ubiquitin was eluted at 45% of the elution buffer. After wards, the fractions were run on a gel to determine the ones containing ubiquitin. The fractions containing ubiquitin were collected together and concentrated to 1 mL using Amicon Ultra filtration flasks (pore size 3 kDa) and its purity was again determined using 15% SDS-PAGE gel. The protein smaple should be as pure as possible.
2.3.3. Concentration determination
The concentration of the protein solution was determined using UV-vis spectroscopy. It was measured at an absorbance of 280 nm (A280) which is the absorbance maxima that aromatic amino acids absorb ultraviolet light. The absorption was measured in a 1 cm cuvette filled with 600 ??l of binding buffer for blanking after which 50 ??l of the sample was added and mixed with the blank. The protein concentration, c, was calculated using the Lambert-Beer law:
A280 = '??c'd where c = concentration [mol L-1]
?? = molar extinction coefficient [L mol-1 cm-1]
d = pathlength [cm]
2.4. Verification of the protein folding using NMR ' I'm not sure of this part!!!
NMR is the only technique that is capable of studying the structure of a protein in solution. The structure of the protein molecule is determined by examining the behaviour of nuclear spins in the presence of a strong external magnetic field and radio wave pulses. With the introduction of the NMR active stable isotopes 13C and 15N, the spins in a protein are almost connected by one-bond couplings which greatly facilitate the study. The most important starting point is the one-bond coupling 1H-15N which is present in every amino acid residue in a protein except the N-terminal and the proline residues.
From the concentrated protein solution, 450 ??l of it was taken and into a 5 mm NMR tube. 50 ??l of deuterium oxide and 2 ??l of DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) were also added to it. The spectrum was recorded on a 700 MHz Bruker Ascend III equipped with a TCI cryoprobe at the NMR Research Centre (EFRE INTERREG IV ETC-AT-CZ programme, Project M00146 "RERI-uasb"). The correlation spectrum 1H, 15N HSQC was recorded and superimposed with an already obtained spectrum of a properly folded ubiquitin.
3.1. Cell Growth and Lysis
Growth was observed in the cell in the LB-agar plates and the bacteria resistant to ampicillin were selected and inoculated into three separate flasks containing 33 ml of the LB media. They were incubated overnight at 37??C and 1 ml was used as a starter culture for the minimal media. The optical densities of the M9 minimal media was measured at OD600 every 30 minutes. Figure 2 shows the growth curve produced before IPTG induction.
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