Size-Exclusion of Dye Molecules
As a demonstration, the instructor may illustrate the concept of size exclusion on a set of mixed food coloring.
Size exclusion chromatography of food coloring.
- Let the column empty over a beaker.
- Carefully load 0.2 ml of food coloring mixture onto the column.
- Place 10 tubes on a rack under the column.
- Place a 1 ml buffer on the column and collect 0.5 ml fractions.
- Continue to add buffer 1 ml at a time until all fractions have been collected.
Size-Exclusion of Proteins
This exercise seeks to purify Green Fluorescent Protein (GFP) or Blue Fluorescent Protein (BFP) from the bacterial lysate. These proteins have a specific size of 238 amino acids and are 40,000 daltons (40kD). Based on their specific size, they will have a specific rate of migration through the size-exclusion resin. Remember that the bacterial lysate is full of additional proteins that are not your protein of interest that we are attempting to isolate.
3D model of GFP (Top Left), BFP (Top Right), structural alignment of GFP and BFP (Center) and the sequence alignment (Bottom) illustrating the 3 amino acid changes to produce the alternative protein. Red asterisks indicate the location of mutations.
Drops of fluid will be collected in fractions. The fractions containing the fluorescent proteins will be found only in specific fractions that will be visible under UV illumination.
- Vertically mount the column on a ring stand. Make sure it is straight.
- Slide the cap onto the spout at the bottom of the column.
- Mix the slurry (molecular sieve) thoroughly by swirling or gently stirring.
- Carefully pipet 2 ml of the mixed slurry into the column by letting it stream down the inside walls of the column.
- Place an empty beaker under the column to collect wash buffer.
- Remove the cap from the bottom of the column and allow the matrix to pack into the column.
- Label eight microcentrifuge tubes #1-8.
- Slowly load the column with 0.2ml of the GFP extract. Allow the extract to completely enter the column.
- Add 1ml of the elution buffer on top of resin without disturbing the resin.
- Add buffer slowly (several drops at a time) to avoid diluting the protein sample.
- Using the graduated marks on the sides of the tubes, collect 0.5ml fractions in the labeled microcentrifuge tubes.
- Continue to add 1ml buffer and collect fractions until all tubes are full.
- Check all fractions by using long wave U.V. light to identify tubes that contain the fluorescent GFP or BFP proteins.
- Further purification may be performed with a different resin with the few fractions containing the protein of interest.
- Protein samples should be run on an acrylamide gel and stained against all proteins to check the purity of the sample or fluorescence measurements taken.
Isolation, purification, gene cloning and expression of antifungal protein from Bacillus amyloliquefaciens MG-3
Antifungal protein was isolated and purified from B. amyloliquefaciens MG-3.
Antifungal protein is a serine protease with a molecular weight of
Antifungal protein showed good stabilities to temperature, pH and protease K.
Antifungal protein inhibited pathogenic fungi growth and fruit decay in loquats.
Recombinant antifungal protein effectively suppressed the growth of C. acutatum.
CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag
There are many strategies to purify recombinant proteins of interest, and affinity purification utilizing monoclonal antibody that targets a linear epitope sequence is one of the essential techniques used in current biochemistry and structural biology. Here we introduce a new protein purification system using a very short CP5 tag. First, we selected anti-dopamine receptor D1 (DRD1) rabbit monoclonal antibody clone Ra62 (Ra62 antibody) as capture antibody, and identified its minimal epitope sequence as a 5-amino-acid sequence at C-terminal of DRD1 (GQHPT-COOH, D1CE sequence). We found that single amino acid substitution in D1CE sequence (GQHVT-COOH) increased dissociation rate up to 10-fold, and named the designed epitope sequence CP5 tag. Using Ra62 antibody and 2 peptides with different affinity, we developed a new affinity protein purification method, CP5 system. Ra62 antibody quickly captures CP5-tagged target protein, and captured CP5-tagged protein was eluted by competing with higher affinity D1CE peptide. By taking the difference of the affinity between D1CE and CP5, sharp elution under mild condition was achieved. Using CP5 system, we successfully purified deubiquitinase CYLD and E3 ubiquitin ligase MARCH3, and detected their catalytic activity. As to G protein-coupled receptors (GPCRs), 9 out of 12 cell-free synthesized ones were purified, demonstrating its purification capability of integral membrane proteins. CP5 tagged CHRM2 expressed by baculovirus-insect cell was also successfully purified by CP5 system. CP5 system offers several distinct advantages in addition to its specificity and elution performance. CP5 tag is easy to construct and handle because of its short length, which has less effect on protein characters. Mild elution of CP5 system is particulaly suitable for preparing delicate proteins such as enzymes and membrane proteins. Our data demonstrate that CP5 system provides a new promising option in protein sample preparation with high yield, purity and activity for downstream applications in functional and structural analysis.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
2. Materials and methods
2.1. Camel small intestine
Camel small intestine was obtained from Cairo slaughter house. The small intestine was saved directly into an ice box for transportation to the laboratory and transferred to frozen storage at ଌ.
2.2. Deoxyribonuclease assay
Deoxyribonuclease (DNase) activity measurements were carried out according to Yasuda et al. . The one ml reaction mixture consisted of 20 µg calf thymus DNA, 10 mM MgCl2, 10 CaCl2, 50 mM Tris-HCl buffer, pH 7.0 and 2 µg protein. The change in absorbance at 260 nm was followed at 30 s intervals. One unit of DNase activity was defined as the amount of enzyme which increases the O.D. 1.0 per min under standard assay conditions.
2.3. Purification of DNase from camel small intestine
2.3.1. Preparation of crude extract
The DNase crude extract was prepared by homogenization of 5 g camel small intestine in 15 ml of 20 mM Tris–HCl buffer, pH 7.0 using a homogenizer. The homogenate was centrifuged at 10,000 g and the supernatant was designated as crude extract. The crude extract was subjected to dialysis against the same buffer.
2.3.2. Column chromatography
The dialyzate was applied directly to a diethylaminoethanol (DEAE)-Sepharose column (10 ×ਁ.6m i.d.) pre-equilibrated with the same buffer. The adsorbed material was eluted with a stepwise gradient of NaCl ranging from 0.0 to 0.2 M prepared in the same buffer at a flow rate of 30 ml/h and 3-ml fractions were collected. The pooled fractions (0.2 M NaCl) with the highest specific activity of DNase were concentrated through dialysis against solid sucrose and applied on a Sephacryl S-200 column (90 ×ਁ.6m i.d.) previously equilibrated with the same buffer and developed at a flow rate of 20 ml/h, and 3.0 ml fractions were collected.
2.4. Protein determination
Protein was quantified by the method of Bradford . Bovine serum albumin was used as the protein standard.
2.5. Molecular weight determination
Molecular weight was determined by gel filtration technique using Sephacryl S-200. The column (90 ×ਁ.6m i.d.) was calibrated with cytochrome C (12,400), carbonic anhydrase (29,000), bovine serum albumin (67,000), alcohol dehydrogenase (150,000) and β-amylase (200,000). Dextran blue (2,000,000) was used to determine the void volume (Vo). Subunit molecular weight was estimated by SDS-polyacrylamide gel electrophoresis . SDS-denatured phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,000) and α-lactalbumin (14,200) were used for the calibration curve.
2.6. Characterization of DNase 3
The effect of pH on DNase 3 activity was determined in the pH range from 4.5 to 9.0 using 50 mM sodium acetate buffer (pH 4.5𠄶.0), sodium phosphate buffer (pH 6.5𠄷.5) and Tris-HCl buffer (pH 7.0𠄹.0). The optimal temperature for DNase activity was determined by incubating the enzyme-substrate mixtures at various temperatures (10 ଌ) in 50 mM Tris-HCl buffer, pH 7.0. Thermal stability of DNase was measured in terms of residual activity after incubation of DNase at different temperatures (10 ଌ) for 1 h prior to substrate addition. The Km value was determined from Lineweaver-Burk plot using DNA concentrations from 20 to 100 µg. The effect of metal cations on DNase activity was investigated by preincubating the enzyme with 10 mM Mg 2+ , Ca 2+ , Zn 2+ , Co 2+ , Hg 2+ , Ba + , Cd 2+ and Ni 2+ for one h prior to substrate addition.
Antibody Purification using Protein A, Protein G, or Protein L Agarose
This protocol is designed as a quick purification method for antibodies from mammalian sera, ascites, and cell culture supernatants. It should be noted that if the starting material is serum or ascites the final preparation will contain endogenous host IgG as well as specific antibodies. In general, the presence of this endogenous IgG should not interfere with assays using the antibodies. The immunoglobulin content of normal sera from several species and from monoclonal antibody sources are given in Table (PDF).
Note: We offer the PURE1A Kit for purification of antibodies using protein A.
Purification Protocol (for a 1 mL column)
Notes: This protocol uses a high molarity, high pH loading buffer for Protein A to enhance binding of subclasses with a weak affinity for Protein A (such as mouse IgG1). Binding to Protein G and L takes place at neutral pH, so phosphate buffered saline is used as the loading buffer. Elution of bound Ig is accomplished in a single step with a citrate buffer, pH 3, which removes all subclasses that have bound to the resin. The capacity of Protein A and Protein G for IgG from various species may be estimated from the relative affinities given in Table (PDF). An IgG subclass with 1-2 pluses will bind at 1-5 mg IgG per mL resin. A subclass with 3-4 pluses will bind at 10-25 mg per mL resin. Protein L is able to bind all Ig classes and can bind 3-10 mg Ig per mL resin for species with 4 pluses. It is recommended that the resin to be used have at least 2 pluses for the species and Ig class of the material to be purified.
Reagents and Equipment
- Serum, ascites or cell culture supernatant
- Protein A Loading Buffer: 1 M potassium phosphate, pH 9.0
- Protein G or L Loading Buffer: 0.01M phosphate buffered saline (PBS), pH 7.4 (Product No. P4417)
- Elution Buffer: 0.1 M citric acid, pH 3.0
- 1.5 M Tris base, for neutralization of the eluate
- 3 - 5 mL syringe (column sleeve), glass wool
- Stopcock, Luer-Lock (Product No. S7396)
- Ring stand, clamp
- Test tubes, rack
- Spectrophotometer, cuvets
- pH meter
- Transfer pipettes, bulbs
- Beakers, stirbars and stirplate (for buffer preparation)
- Sterile filter units (optional)
- Sodium azide (preservative) (Product No. S2002)
- Prepare buffers. Buffers may be stored at 4 °C for 1-2 weeks. Filtration through sterile filter units will prolong the life of the buffers, but is not required.
- Prepare column sleeve. Remove plunger from syringe and discard. Press a small amount of glass wool into the bottom of the syringe, enough to form a cushion about 1/2 - 1 cm thick. Attach the stopcock. Rinse with 1-2 mL loading buffer. Ensure that the glass wool cushion remains firmly in the bottom of the syringe.
- Suspend the resin in 2 mL loading buffer by inverting and rotating the bottle. Avoid excessive shaking. Do not vortex the slurry.
- Pour the slurry into the syringe. Allow the excess buffer to drain through, then wash the column with 5 mL loading buffer. Do not allow the resin to run completely dry.
- If possible, estimate the amount of Ig in the serum, ascites, or supernatant to be loaded. If the amount of Ig is not known, Table (PDF) contains approximate levels of Igs in serum from different species, in mouse ascites, and in cell culture supernatant that may be used to estimate the amount of Ig in the starting material. Based on the capacity of the resin for Ig from the species of the starting material (see Notes above) and the amount of Ig in the starting material, calculate the volume to load.
Note: These are approximate values, to be used only as an initial guide. Actual Ig concentrations in fluids and binding to the resins will vary and optimal ratios of starting material to resin must be determined experimentally.
Protein Purification is a free online program developed by the late (June 2016) Dr. Andrew G. Booth at the University of Leeds (http://www.agbooth.com/pp_ajax/) 1 . The program offers four protein mixtures of varied complexity to explore: Easy3 (three proteins), Example (six proteins), Default (20 proteins), and Complex (60 proteins). Users can select any of the proteins within a mixture to purify. The separation techniques available are ammonium sulfate fractionation, heat treatment, gel filtration, ion exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. Within each of these techniques, users can alter various parameters. In the case of ion exchange chromatography, anion-exchange media (Q-Sepharose and DEAE-cellulose) or cation-exchange media (S-Sepharose and CM-cellulose) are available. With either, users define the method of elution as either a “salt gradient” or “pH gradient”. With salt gradient selected, users can define the pH of the equilibration buffer (2.0–11.0) and the start and end salt concentrations within a 0 to 3.0 m range. When a column-based purification technique is chosen, a FPLC chromatogram appears that displays absorbance at 280 nm (A280) on the primary y-axis, fraction number on the x-axis, and [salt] on the secondary y-axis (see Fig. 1). Although not employed in our exercise, the program also features virtual 1D- and 2D-gels (with coomassie blue and immunoblot staining available) that enable users to analyze fractions from a column. The program also contains a help feature that includes six tutorial exercises, but practice with these is not required for the exercise described in this work. A literature search indicates that two publications using the program are available, and both are from the author of the program 1, 4 . More recently, a web-based applet that simulates an ion exchange purification of over produced proteins from E.coli was described 5 . However, we decided to use the program devised by Dr. Andrew Booth for its ease of use and versatility that extends to techniques beyond ion exchange chromatography 1, 4 .
Screen shots of FPLC chromatograms using the program. Each simulation used the “Easy3” mixture. Asterisks indicate the location of Protein 1 with the estimated [salt] required for elution given. Ion-exchange media, equilibration pH, and the salt gradient varied as follows:
(A) Q-Sepharose, pH 6.0, 0–0.5 m salt, (B) Q-Sepharose, pH 7.0, 0–0.5 m salt, (C) Q-Sepharose, pH 8.0, 0–0.5 m salt, (D) Q-Sepharose, pH 10.0, 0–0.5 m salt, (E) Q-Sepharose, pH 10.0, 0–1.0 m salt, (F) DEAE-Cellulose, pH 10.0, 0–0.5 m salt. Panels lettered according to the exercise (see Supporting Information, page 3). [Color figure can be viewed at wileyonlinelibrary.com]
The purification of a protein is the essential initial step in the study of its physical and biological properties and is one of the most common procedures in biochemistry. This article describes a method for teaching purification skills through the partial isolation of ferredoxin-NADP + reductase and ferredoxin from a single cell batch. The method has been used for several years in an introductory biochemistry course using spinach leaves as cellular source. The protocol gives a complete picture of the preparation of a crude extract and the subsequent isolation of both electron transport proteins on a laboratory scale. It introduces students to the use of different techniques for the purification and detection of proteins and allows them to develop a number of valuable experimental and analytical skills without necessarily resorting to complicated or expensive equipment.
Laboratory experience helps students to develop their critical thinking and creativity. Moreover, by doing practical work they increase their appreciation of the mechanisms by which scientists obtain and analyze information. It is actually through the laboratory training that the students become intensely and personally involved in the acquisition of knowledge. Based on this idea, we have developed in the last years a short practical training on protein purification as part of a general biochemistry course.
Biochemical work frequently requires the purification of a particular compound from a complex mixture. In fact, proteins constitute one of the probable candidates to be handled or isolated by the biochemistry student along its professional carrier. As a model for developing this skill we have focused our interest on the purification of ferredoxin-NADP + reductase (FNR) 1 1 The abbreviations used are: FNR, ferredoxin-NADP + reductase DCPIP, 2,6-dichlorophenolindophenol. from spinach, which in addition allows the obtainment of the protein ferredoxin as a side product. Both proteins are colored (FNR is yellow, and ferredoxin is brown), relatively abundant in the starting material, easy to handle, and present pronounced biochemical differences, which enable students to make interesting comparisons.
FNR is the FAD-containing enzyme responsible for NADP + photoreduction in plants, green algae, and cyanobacteria [ 1 ]. It has been isolated from many sources and extensively characterized. The molecular mass of FNR from spinach is 36,000 Da, and its isoelectric point is around 5. The ability of FNR to reduce artificial substrates such as 2,6-dichlorophenolindophenol (DCPIP) or ferricyanide (diaphorase activity) using NADH or NADPH as electron source [ 2 ] allows easy recognition of this enzyme in any stage of purification.
Ferredoxin is a small iron-sulfur electron carrier protein that performs the transfer of electrons from photosystem I to FNR and is involved in many other biological electron transport chains in cyanobacteria and plants and is widely distributed in bacteria also [ 3 ].
Because of the special characteristics of both proteins and their simultaneous presence in the spinach leaves, a starting material that is cheap and readily available all over the world, we consider this is an excellent system for teaching the principles of protein purification. In the practice, the student is instructed in the logical process of purification, which starts from a raw material by applying some preparatory steps of purification. Later the purification is refined, and the purity of the product obtained is evaluated. At the same time, students are trained in some important biochemical aspects such as (a) the study of the spectral properties of a protein, (b) the determination of the time course of product formation in an enzyme-catalyzed assay with calculation of activity units, (c) the measurement of protein concentration based on values obtained from indirect data using different dilutions, (d) the determination of a protein molecular weight by SDS-polyacrylamide gel electrophoresis, or (e) the elaboration of a written report.
This practice has been designed to be performed in a short time, and the protein preparation obtained is only partially purified. However, we explain that in preparative separations, the main aim is to isolate and recover an amount as large as possible of the compound in a high degree of purity to subsequently study its chemistry and/or its biological properties. Students also learn that any purification procedure adopted will inevitably involve some loss of material and that it is essential that the number of purification steps should be kept to a minimum, and therefore the techniques used should be those that are capable of giving the greatest purification yield.
To provide the students a basic background for a better understanding of this practice, it is preceded by a short theoretical training on the principles of protein purification. Moreover, students are encouraged to read and learn more about protein purification methods and analysis in the biochemistry lab books available at the university. We mainly use the texts of Wilson and Walker [ 4 ] and Boyer [ 5 ]. The development of this practical work has shown to be very useful to pose questions whose finality is to encourage students to think about what is observed and to stimulate the imagination of the student. We have verified that this constitutes a good method for a didactic teaching of sciences.
Protein Expression and Purification Core Facility
The Protein Production and Purification Facility is a core service and resource to overcome the major bottleneck in recombinant protein expression and purification. Mainly we provide advice and active support for researchers scientific community in the use of the procaryotic and eukaryotic expression system for recombinant protein production.
The facility offers equipment and materials required for the expression of protein in bacterial, Yeast, insect (baculovirus) and mammalian cell. We can execute the complete cloning, expression, scale-up and purification process or specific tasks within the project.
We have optimized different expressions vectors in combination with various bacterial strains, yeast strain, insect strain and mammalian cell for specific expression of different types of proteins.
The Protein Expression Facility is a fee-for platform dedicated to providing technical assistance in the following areas:
- Advices and training
- Recombinant DNA engineering
- Recombinant protein expression in bacteria and yeast
- Recombinant protein expression via baculovirus expression systems (BVES)
- Recombinant protein expression in Mammalian cells
- Protein purification
- Polyclonals antibodies production
The platform possesses expertise to produce and to purify any type of protein. For each project we used the following flowchart:
- Primer design and expression vector cloning
- Strains development
- Expression screening
- Production and purification
- Preparation and distribution of recombinant expression vector
- New Vector development to provide state-of-the-art expression technologies
- Maintenance of frozen stocks of recombinant baculovirus
- Protein purification technology development
- Development of recombinant protein and molecular biology enzymes catalogue
Fermenter for cell culture and protein production
Chromatography system: for protein purification
Cell Disruption: proteins extraction
Insect cell culture for protein production
Hybridoma system production
Akta flux for sample concentration
Network and collaboration
P4EU: (Protein Production and Purification Partnership in Europe) network initiative
Collaboration with Recombinant Platform facility of Institut Pasteur
The facility functions as a training centre for students and scientists in gene cloning and Recombinant protein production.
Protein Purification Strategies
Proteins are biological macromolecules that maintain the structural and functional integrity of the cell, and many diseases are associated with protein malfunction. Protein purification is a fundamental step for analyzing individual proteins and protein complexes and identifying interactions with other proteins, DNA or RNA. A variety of protein purification strategies exist to address desired scale, throughput and downstream applications. The optimal approach often must be determined empirically.
The best protein purification protocol depends not only on the protein being purified but also on many other factors such as the cell used to express the recombinant protein (e.g., prokaryotic versus eukaryotic cells). Escherichia coli remains the first choice of many researchers for producing recombinant proteins due to ease of use, rapid cell growth and low cost of culturing. Proteins expressed in E. coli can be purified in relatively high quantities, but these proteins, especially eukaryotic proteins, may not exhibit proper protein activity or folding. Cultured mammalian cells might offer a better option for producing properly folded and functional mammalian proteins with appropriate post-translational modifications (Geisse et al. 1996). However, the low expression levels of recombinant proteins in cultured mammalian cells presents a challenge for their purification. As a result, attaining satisfactory yield and purity depends on highly selective and efficient capture of these proteins from the crude cell lysates.
To simplify purification, affinity purification tags can be fused to a recombinant protein of interest (Nilsson et al. 1997). Common fusion tags are polypeptides, small proteins or enzymes added to the N- or C-terminus of a recombinant protein. The biochemical features of different tags influence the stability, solubility and expression of proteins to which they are attached (Stevens et al. 2001). Using expression vectors that include a fusion tag facilitates recombinant protein purification.
Isolation of Protein Complexes
A major objective in proteomics is the elucidation of protein function and organization of the complex networks that are responsible for key cellular processes. Analysis of protein:protein interactions can provide valuable insight into the cell signaling cascades involved in these processes, and analysis of protein:nucleic acid interactions often reveals important information about biological processes such as mRNA regulation, chromosomal remodeling and transcription. For example, transcription factors play an important role in regulating transcription by binding to specific recognition sites on the chromosome, often at a gene’s promoter, and interacting with other proteins in the nucleus. This regulation is required for cell viability, differentiation and growth (Mankan et al. 2009 Gosh et al. 1998).
Analysis of protein:protein interactions often requires straightforward methods for immobilizing proteins on solid surfaces in proper orientations without disrupting protein structure or function. This immobilization must not interfere with the binding capacity and can be achieved through the use of affinity tags. Immobilization of proteins on chips is a popular approach to analyze protein:DNA and protein:protein interactions and identify components of protein complexes (Hall et al. 2004 Hall et al. 2007 Hudson and Snyder, 2006). Functional protein microarrays normally contain full-length functional proteins or protein domains bound to a solid surface. Fluorescently labeled DNA is used to probe the array and identify proteins that bind to the specific probe. Protein microarrays provide a method for high-throughput identification of protein:DNA interactions. Immobilized proteins also can be used in protein pull-down assays to isolate protein binding partners in vivo (mammalian cells) or in vitro. Other downstream applications such as mass spectrometry do not require protein immobilization to identify protein partners and individual components of protein complexes.
Purification, redox sensitivity, and RNA binding properties of SECIS-binding protein 2, a protein involved in selenoprotein biosynthesis
In mammalian selenoprotein mRNAs, the highly structured 3' UTR contains selenocysteine insertion sequence (SECIS) elements that are required for the recognition of UGA as the selenocysteine codon. Our previous work demonstrated a tight correlation between codon-specific translational read-through and the activity of a 120-kDa RNA-binding protein that interacted specifically with the SECIS element in the phospholipid hydroperoxide glutathione peroxidase mRNA. This study reports the RNA binding and biochemical properties of this protein, SECIS-binding protein 2 (SBP2). We detected SBP2 binding activity in liver, hepatoma cell, and testis extracts from which SBP2 has been purified by anion exchange and RNA affinity chromatography. This scheme has allowed us to identify a 120-kDa polypeptide that co-elutes with SBP2 binding activity from wild-type but not mutant RNA affinity columns. A characterization of SBP2 biochemical properties reveals that SBP2 binding is sensitive to oxidation and the presence of heparin, rRNA, and poly(G). SBP2 activity elutes with a molecular mass of approximately 500 kDa during gel filtration chromatography, suggesting the existence of a large functional complex. Direct cross-linking and competition experiments demonstrate that the minimal phospholipid hydroperoxide glutathione peroxidase 3' UTR binding site is between 82 and 102 nucleotides, which correlates with the minimal sequence necessary for translational read-through. SBP2 also interacts specifically with the minimally functional 3' UTR of another selenoprotein mRNA, deiodinase 1.