Identification, production and use of polyhydroxy-reactive monoclonal antibodies - for immunoaffinity chromatography
Identification, production and use of polyhydroxy-reactive monoclonal antibodies - for immunoaffinity chromatography
All forms of affinity chromatography require the formation of a specific interaction between two components by which one of the components can be purified. Immunoaffinity chromatography, which utilizes the specific interaction of antigen and antibody, is a classification that follows the principles of affinity chromatography [for review, see Subramanian (2002)]. In fact, immunoaffinity chromatography is an extension of the scale-up of immunoprecipitation procedures, with the difference that a component (usually the antigen), i.e. the active protein, needs to be recovered after chromatography.
Operation method
Identification, production and use of polyhydroxy-reactive monoclonal antibodies - for immunoaffinity chromatography Move I. Polyhydroxy reactive monoclonal antibodies We were the first to use a special class of monoclonal antibodies and to use them in immunoaffinity chromatography. These antibodies can be used for mild immunoaffinity chromatography because the elution conditions require only a combination of non-ionizing salts and low molecular mass polyhydroxylated compounds (polyols), and the proteins are non-denatured under these conditions. We categorize this antibody as a polyhydroxy-reactive monoclonal antibody (PR-mAb). Our laboratory studies protein transcription. Therefore, most of the PRmAb that we isolate are transcriptionally related mAb in either prokaryotic or eukaryotic systems. eukaryotic transcription systems present a significant challenge for scientists who specialize in isolation, because many factors are actually multisubunit proteins. For example, eukaryotic RNA polymerase n (RNAPn) contains 12 subunits (12 different gene products). However, to initiate transcription from a promoter, RNAPn also requires the transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH [for review, see Woychik and Hampsey (2002)]. With the exception of TFIffi, all of these transcription factors are composed of two or more subunits. Large protein complexes are ideal for immunoaffinity chromatography using PR-mAb. We have developed PR-mAb for E. coli RNAPCThompsonetal.,1992) and eukaryotic RNAPII (Thompsonetal.,1990), as well as PR-mAb for some eukaryotic transfer factors (Table 28.1). Most notable in the application of PR-mAb technology, yeast RNAPIKEdwardsetal.,1990) purified using our PR-mAb8WG16 has been used for protein complex crystallization (Crameretal.,2000). Various PR-mAb have been successfully isolated by other laboratories and applied to purify target proteins (Jiangetal., 1995; Lynchetal., 1996; Nagyetal., 2002). The identified mAb can also be screened for use in polyhydroxylation reactions. Several reviews of PR-mAb have been published (BurgessandThompson,2002;Thomp-sonandBurgess,1996,2 0 01;Thompsonetal.,2 00 6). In this chapter, we will update some of these methods. We will also describe a method for preparing large quantities of RP-mAb using a very inexpensive cell culture system, as well as the preparation of very small amounts of mAb from mouse ascites in the same manner.Finally, based on the polyhydroxylation reaction characteristic of the antibody, the epitope tagging of an unrelated target protein with an RP-mAb by recombinant DNA can be used to purify the protein by a mild polyhydroxylation-elution method. (1) By screening a large number of monoclonal antibodies (218 antigen-specific wells), we expect PR-mAb to represent 5% to 10% of monoclonal antibodies (Thompsonetal., 1992). (2) Screening at the master-well stage allows immediate identification of PR-mAb. (3) PR-mAb is not restricted by mouse IgG subtype. PR-mAb has also been identified and screened in rat mAb (R.R. Burgess, unpublished data). (4) Most PR-mAb are affected by different combinations of salts and polyhydroxy compounds. The most common were 0.75mol/L ammonium sulfate or 0.75mol/L sodium chloride in combination with 30%~40% propylene glycol. (5) PR-mAb can be a high affinity antibody. In fact, in diluted solution, high affinity is the first condition to ensure effective binding of mAb to antigen. It becomes a low affinity antibody when eluted. (6) Most (not all) PR-mAb are affected by different combinations of salts and polyhydroxylated compounds. Salts have been verified to include ammonium sulfate, sodium chloride, sodium acetate, and potassium glutamate! Polyols include propylene glycol, ethylene glycol, 2,3-butanediol, and sometimes glycerol. (7) Salts and multisubunit compounds are commonly used as protein stabilizers, whose mild elution preserves the biological properties of the antigen and its structural integrity, and can even be used in multisubunit complexes (CrameretaL, 2000; Thomp.sonetal., 1990). We have used the hybridoma technique to prepare mAb (Harl o wandLane, 1988), i.e., the fusion of antigen-stimulated mouse splenocytes with a myeloma cell line. It has been demonstrated that highly immunized mice should be preferred; only 5%-10% of the monoclonal antibodies are PR-mAb, and therefore a large number of primary hybridoma cells are required to isolate PR-mAb. However, mAb can be prepared using other methods, such as plasma cells infected with a retrovirus (Largaespadaetal., 1"6), or antibody libraries can be constructed by recombinant techniques. The mAb can be prepared Regardless of the source, the antibody must be antigen-specific. We found that a standard (ELISA) was the way to go. We then screened the antibodies using a modified ELISA, the ELISA-elution assay. This method is based on the standard ELISA with an additional step of co-treating the specific antigen-antibody complex with salt and polyhydroxylated compounds prior to the addition of the enzyme-linked secondary antibody. The general procedure of the ELISA-elution assay is described below. (1) Antigen-coated polystyrene microtiter plate. Typically 50 wells of phosphate buffer solution (PBS, pH 7.4) containing 30-100ng of antigen are added to each well. Incubate at room temperature for Ih to ensure sufficient time for the antigen to bind to the polystyrene. (2) Each well is blocked with 200 buckets of PBSd%BLOTTO) containing 1% skimmed milk powder. Usually overnight at 4°C, 2 h at room temperature is sufficient. (3) Add the antibody to be measured (50(uL)) to two adjacent wells and incubate at room temperature for Ih. Normally, the antibody in the cell culture medium can be used directly D. However, antibodies with very high affinity or very high titers should be diluted to a non-saturating level for use. (4) Wash the plate five times with PBS (PBST) containing 0.l% tween20 to remove unbound antibodies. (5) For control wells, add 100 fJLTE buffer [50 mmol/L Tris-HCl (pH 7.9), 0.1 mmol/LEDTA] to each well, and for the wells to be tested, add 100 TE buffer containing 0.75mol/L ammonium sulfate and 40% propylene glycol to each well. The EUSA plate was incubated at room temperature for 20 min, and the side of the ELISA plate was tapped occasionally (approximately every 5 min) to mix the solution. (6) Wash the plate 5 times with PBST. (7) Dilute commercial horseradish peroxidase-labeled secondary antibody (usually 1:2000) with 1% BLOTTO and add 50 |uL per well. incubate at room temperature for Ih. (8) Wash the plate 10 times with PBST. (9) Add appropriate substrate to the wells. We used 100^ of 0.05 mol/L citrate buffer (pH 5.0) containing 0.03% H2O2 and 0.4 mg/mL o-phenylenediamine (OPD). (10) Enzyme-linked plates were incubated at room temperature for 5 to 15 min. reactions were terminated in pairs (TE and TE+salt/polyol for each monoclonal antibody) with the addition of 50 fxLImol/LH2SO4 per well. (11) Absorbance values were read by enzyme coupler. For ()PD, the detection wavelength is 490 nm. The absorbance value of PR-mAb in the wells to be tested treated with polyols and salts will be reduced by approximately 50% compared to the control wells with TE buffer only (Fig. 28.1A). If an enzyme coupler is not available, a decrease in absorbance of approximately 50% is usually apparent by visual inspection. (1) PR-mAb Screening can be performed at the master-well stage. Hybridoma cell screening is used for specific antibody preparation, followed immediately by PR-tnAb screening. Initial screening can be performed in 100 cell cultures (50 control buffer and 50;^ buffer containing polyhydroxy compounds and salts) (Thompsonetal., 1992). In one study, we screened more than 200 hybridoma cells for PR-mAb preparation in a single fusion at the primary well stage. (2) Antigen binding to a microtiter plate can lead to changes in antigen structure. This may result in the exposure of epitopes buried within proteins in solution, leading to false positives because the mAb does not react with the target in solution, and this mAb is not suitable for immunoaffinity chromatography. (3) When several milliliters of cell culture solution are available, the effect of different concentrations of polyols and salts on the mAb can be examined on a single plate (Fig. 28.1B). In many cases, the practice of producing mouse mAb from ascites is no longer encouraged. Therefore, alternative methods have been developed for the production of large quantities of mAb for immunoaffinity chromatography, and this section describes a method of antibody preparation that has proven to be well suited for use on this scale. In this procedure, a commercial CELLineFlask350 (CL350) cell culture chamber from IntegraBiosciencesAG (Switzerland) is used. This product is also available from ArgosTechnologies (Elgin, IL) and Bioraco International (Framingham, MA). We have found this product to be easy to use, capable of preparing 10-50 mg of antibody, and the same hybridoma cells can be reused several times. Requirements include standard aseptic cell culture techniques, a cellculturehood, and a WC humidified CO2 incubator (5%). A schematic diagram of the culture flask is shown in 28.2A. Routine procedures are described below. (1) Preparation of Cell Culture Media. Two slightly different media are prepared for use in two CL350 dishes. Dulbecco's Modified Eagle Medium [Dulbecco's modifiedeaglemedium (DMEM)] contains glutamine and a high concentration of dextrose (Gibco/Invitrogen #11965) with the addition of Immol/L pyruvic acid na (Sigma), 100 units/mL penicillin, 100 fxg/mL streptomycin (Gibco/Invitrogen), and 100 fxg/mL streptomycin (Sigma). mL penicillin, and 100 fxg/mL streptomycin (Gibco/Invitrogen) as the base components of both culture media. In the cell growth chamber, DEME requires the addition of these components and 15% inactivated fetal bovine serum (Hycl0ne). In the nutrient maintenance chamber, DMEM requires the addition of the above ingredients and 5% fetal bovine serum to the DMEM culture medium. We call the medium for the cell growth chamber "completeemedium" and the medium for the nutrient maintenance chamber "nutrientmedium". (2) Prepare the inoculum for inoculating the culture bottles. Remove a tube of hybridoma cells from the liquid nitrogen tank and thaw it quickly at 37°C. Hybridoma cells were plated in complete medium at a density of approximately 2XIO4 cells/mL. We used 10 cm cell culture plates containing 20 mL of medium. (3) Inoculate culture flasks. Prepare 5 mL of fresh complete medium containing 8XIO6~20XIO6 live cells in logarithmic growth phase. Add 25 mL of Nutrient Medium to the Nutrient Maintenance Chamber (green lid) to moisten the membrane between the Nutrient Maintenance Chamber and the Cell Growth Chamber before cells are spread in the Cell Growth Chamber. Suspend cells and pipette out with 10 mL of serum. Unscrew the green cap, insert the pipette securely into the cell growth chamber, and inoculate 5 mL of suspension into the cell growth chamber (white cap). Remove air bubbles by slowly blowing the liquid up and down, allowing them to rise before blowing the liquid back into the chamber. Replace the white cap and tighten. Add 35 0 mL of nutrient medium to the nutrient maintenance chamber and tighten the green cap. (4) Harvest cell growth chamber and culture maintenance. Replace the nutrient medium in the nutrient maintenance chamber every 3-7 days. Unscrew the green cap and insert a 10 mL pipette into the cell growth chamber, blowing the liquid up and down to thoroughly mix the cells. Then transfer the entire liquid in the cell growth chamber to a centrifuge tube. Due to osmotic flux, the volume may be greater than 5 mL. After sampling, cells are counted by hemocytometry and assayed by live cell staining with Taipan blue. Remove the contents of the cell growth chamber and centrifuge to precipitate the cells. Remove the combined rnAb-containing medium and freeze the supernatant for later purification. Resuspend the cells in fresh complete medium. It may be necessary to divide the cells into several portions (usually 1:2 to 1:4) depending on the initial inoculum density, growth rate, and frequency of fluid changes. Loosen the green lid and pour 5 mL of cells back into the petri dish (white lid). Remove air bubbles and tighten the white lid. Add 350 mL of medium to the nutrient chamber and tighten the green lid thoroughly. (5) Harvest IntegraCL350 flasks every 3-7 days after the culture is established. The harvest interval depends on the growth rate of hybridoma cells and the ability of hybridoma cells to adapt to the culture bottle environment. This feature is cell line dependent. (1) Culturing hybridoma cells in IntegraCL350 flasks is very effective, with mAb0.5~Img per ml of cell culture harvested supernatant, and can usually be cultured continuously for about 1 month. (2) Handling of liquids: Pre-warm the medium in a 37°C water bath. This helps to avoid condensation in the culture flasks and temperature shock of the cells. When adding or removing liquid to or from the cell culture dishes (white lids), loosen the green lids of the nutrient culture chambers to prevent air from being trapped. Always tighten the white and green caps of the culture flasks before incubating in the incubator. An IOmL serum pipette is recommended. Change the fluid in the nutrient maintenance chamber by pipetting out the medium and adding fresh medium. (3) The minimum cell concentration for inoculation is 1.5X106 cells/mL [step (2)]. We attempted to reduce the amount of fetal bovine serum required in the nutrient medium without success. Some new serum-free media available on the market can be used as nutrient medium. (4) It is important to monitor cell number and status [Step (3)]. It helps to determine whether to isolate cells in order to reduce the number of cells and avoid a total viable cell count greater than I.OXlO8. If cell viability is greatly reduced, the frequency of fluid changes needs to be increased. (5) During continuous culture, the percentage of viable cells [Step (4)] decreases due to cell death caused by isolating cells at each harvest. At the end of the culture period, only 30%~40% of the viable cells are abnormally terminated. (6)-Some hybridoma cells are unstable and lose the ability to produce antibodies when cultured continuously for a long period of time. Therefore, antibody production should be monitored during culture. We used a standard ELISA assay. (7) Information on CELLine culture flasks can be obtained at www.integrabiosciences,COM/celline. In order to maximize binding between the antibody and the available reaction sites on the vector, it is necessary to purify the antibody, and at least partial purification is beneficial. Antibodies can be purified by a number of different methods. One of the most economical and practical methods is molecular exclusion chromatography or ion exchange chromatography. Affinity chromatography using ProteinA, ProteinG, or a combination of the two is commonly used for purification, but this method is costly. Mouse 111 (1) belongs to one of the 04 subclasses of immunoglobulins: ^0 1, ^02&, Turtle 02 dagger, and IgG3. Mouse IgG2a, IgG2b, and IgG3 can be purified by ProteinA chromatography. Mouse IgGl binds poorly to ProteinA. Unlike mouse IgG2a, mouse IgGl binds better to ProteinG, while IgG2b binds to both ProteinA and ProteinG. In our experience, the monoclonal antibodies in most typical fusion cases are of the IgGl subclass. Here we present a DEAE column (WhatmanDE52) that can be used to purify mouse IgGmAb, which is both economical and simple to use. For mAbIgGl, this method is particularly effective. In fact, in our operation, each purified igG1mAb was tested to be about 90% pure. In addition, many mouse IgG2a and IgG2b antibodies can be purified by this method. (1) The antibodies in either ascites or CELLine cell culture supernatant are precipitated with ammonium sulfate. Add saturated ammonium sulfate solution to the mAb to milk % saturation. The slurry is stirred on ice for 20 min. mAb is precipitated in this manner while most of the serum albumin remains in solution. (2) Separate the precipitate by centrifugation (approximately 6000 min, 10 min). Add antibody buffer [50 mmol/LTris-HCKpH6.9), 25 mmol/LNaCl] to the precipitate in the amount of l/4 of the initial volume of antibody (CELLine flask preparation of antibody) and 1/2 (ascites). (3) The precipitate is dissolved in about 15 min and clarified by centrifugation (about 6000 IOmin). (4) Dialyze the supernatant obtained in step (3) overnight at 4°C in IL antibody buffer. (5) The clarified supernatant is used for DE52 column chromatography, and the column is prepared as described in the "Instructions" below. The column is equilibrated with Antibody Buffer (pH 6.9), the pH at which most of the mAb flows through and most of the impurities bind to the column. 10 mL of the raw material can be used on a 5 mLDE52 column. (6) Collect the fractions of the raw material that do not bind to the column (approximately ImL), and perform SD&PAGE on each sample of the fractions. mAb8RB13 (IgGl-type monoclonal antibody) was prepared in CL350 culture flasks, and the SDS^PAGE results of the purified product are shown in Figure 28.2B. The isolated fractions were combined according to antibody purity. (7) Elute the column with 5 mL of antibody buffer containing 0.5 mol/L NaCl, and save the eluate before PAGE analysis. (1) Preparation of DE52 is very important. Although the manufacturer claims that pre-cycling is not necessary, we have demonstrated that pre-cycling greatly improves chromatographic performance. 10 g of resin is suspended in 100 mL of water and washed several times with 100 mL of water, with each wash removing fines (small, non-settling particles). The resin is then treated with 100 mL of 0.Imol/LHCl for 30 min and the HCl is slowly poured off. Wash the resin at least 3 times, each time with 100 mL of water. For the last wash, pour out the liquid slowly, then treat the resin with 0.Imol/LNaOH for 30 min, pour out the liquid slowly, and wash the resin at least 3 times with 100 mL of water each time. Wash the resin 3 times with antibody buffer, each time with 100 mL of buffer, and suspend with the same buffer. Detect pH with pH paper and add sodium azide to a final concentration of 0~0 2%. Dispense the resin into disposable tubes and store in the refrigerator. (2) mAb can be derived from ascites. Although ascites is not pure (80%~90% purity), impurities may not affect the performance of the immunoaffinity resin. (3) Under the above conditions, most mouse mAb can flow through DE52, but a small amount of mAb can still be bound to the chromatography column. Therefore, high salt elution conditions [step (7)] can be used to elute the antibody. Purification of the mAb bound to the DE52 column using a salt gradient concentration is necessary. (4) If the mAb is prepared in CELLine culture flasks, DE52 may not be necessary. A wide variety of resins and coupling agents are available from suppliers. We have validated most of them, but none are more effective than crosslinked agarose derivatized with cyanogen bromide (CNBr). (1) After purification the mAb was dialyzed with coupling buffer (1 00 mm o l/LNaHCO3, 500 mmol/LNaCUpH8.3). The antibody solution is removed from the dialysis tubing and the volume is adjusted with coupling buffer to 10 mL per gram of cyanogen bromide-activated Seph-arose dry powder, and the sample is retained (approximately 1,00^L) for analysis of protein concentration. (2) It takes about 20 min for the cyanogen bromide-activated Sepharose dry powder to swell in 0.Immol/LHC1. 3.5 mL of gel is prepared per gram of cyanogen bromide-activated resin dry powder. The resin is then purged in a glass filter with approximately 100 mL of 0.Immol/LHCl per gram of resin. (3) After a quick wash of the resin with about 20 mL of coupling solution, the resin is transferred to the antibody solution. the gel slurry solution is turned over and mixed for 2 h with a laboratory rotator at 23°C. The gel slurry solution is then mixed with a laboratory rotator for 2 h. The gel slurry solution is then turned over and mixed for 2 h with a laboratory rotator. (4) Collect the resin through a glass filter and save the filtrate for determination of uncoupled protein content. (5) Transfer the resin to a solution of about 1 0 1111^1"[8.3, 1111 0 1/1 ethanolamine,23.(: Turn the mixture over 211 to fully react the ethanolamine with the residual cyanogen bromide. (6) The resin was again collected with a glass filter, coupling buffer (about 50 mL) washed with 100 mmol/L sodium acetate buffer (pH 4.0). (7) Repeat at least 2 or 3 times the 2 washes in step (6). (8) Store coupled Sepharose in I0 mL of coupling buffer containing 0.02% NaN3 at 4°C. (9) Determine the protein content of the antibody solution [sample from steps (1) and (4)] stored before and after binding. The coupling efficiency is determined accordingly. (1) Prepare 3.5 mL of dissolved resin from Ig of dry resin powder. We found that in most cases, it is more convenient to process 0.5~2.0 g of dry resin powder at a time. We found that in most cases, it was easier to process 0.5-2.0 g of dry resin powder at a time, and confirmed that ImL of dissolved resin coupled with 2.5 mgmAb was more effective. Therefore, 3.5 mL(lg) of resin is required to bind 8.75 mgmAb. (2) The pH condition for cyanogen bromide activation is above 8. Therefore, the antibody solution in step (3) above should be transferred to the resin as soon as possible. (3) The blocking agent [Step (5)] may vary depending on the purpose of use. For example, an ethanolamine-binding protein from yeast will be co-purified with the target protein if ethanolamine is used as a blocking agent. In this case, almol/L glycine is a suitable blocker. (4) Store the resin in 0. 0 2% NaN3 at 4°C. The resin can be stored stably at this condition. Under this condition, the resin can be stored stably for about 6 months. It has been noted that some antibodies will peel after 6 months of storage. Adding 50% glycerol to the above buffer and storing at 20°C (not frozen) may extend the half-life. In the following, we use mAbNT73 or 8RB13 to purify RNA polymerase of E. coli origin as an example. The one-step immunoaffinity chromatography method described earlier yields an RNA polymerase that is about 90% pure. A number of other proteins that bind to the RNA polymerase will also be co-eluted. The procedure is based on obtaining an E. coli precipitate (2 to 3 g wet weight) from an IL logarithmic end-of-growth culture. The SD^PAGE results shown in Figure 28.3 are the results of purification using this method. (1) The precipitate can be partially re-melted on ice and resuspended in 20 mLTEN buffer [50 mmol/LTris-HCl (pH 7.9), 0.Immol/LTris-LTA, 100 mmol/LNaCl]. (2) Add lysozyme to a final concentration of 250 pg/mL, and incubate cells on ice for 20 min. or use 1500 kU recombinant lysozyme (EMD/Novagen #H110). (3) Sonicate the cells on ice, repeat 4 times, 15 s each time with 15 s intervals. (4) Centrifuge the lysate at 15000r/min (27000 g) for 15 min. (5) Sample the supernatant (1~2 mL) onto an immunoaffinity column at 23°C and collect the flow-through fraction. (6) Wash the column with TE (approx. 20 mL) containing 100 mmol/L NaCl and then TE (approx. 5 mL) containing 500 mmol/L NaCl. The column was re-equilibrated with TE (approx. 10 mL) containing 100 mmol/L NaCI. (7) Elute the column with TE containing 0.75 mol/L NaCl and 40% propylene glycol (room temperature). Collect the eluted fractions on ice. (8) SDS-PAGE electrophoresis to separate the eluted peaks. The SDS-PAGE results in Figure 28.3 show the purity of the one-step chromatography product. Combine the desired fractions (Fig. 28.3, lanes 5 and 6) and dialyze with a suitable storage buffer [for transcriptional proteins, 50 mmol/LTris-HCl (pH 7.9), 50 mmol/LNaCUO.Immol/LEDTA.O.lmmol/LDTT, and 20% to 50% glycerol]. (1) Treat the lysate [step (3)] with Benzonase nuclease (EMD/Novagen#7 0 746, Madison,WD, which helps to digest the nucleic acids and reduce the viscosity (see Chapter 18 of this book). (2) A 500 mmol/L NaCl wash [Step (6)] helps eliminate nucleic acids and NusA, which is an RNA polymerase binding protein. (3) Elution of the target protein is more efficient at room temperature than at 4°C [Step (8)] for unknown reasons. Proteins or protein complexes can often be purified from biological materials that are not genetically engineered. It has also been found that the use of PR-mAb's that recognize highly conserved epitopes can lead to greater applicability of immunosorbents. Two of the most successful PR-mAb applications can cross-react with the same enzyme in many species. mAb8WG16 interacts with a repeating heptapeptide at the C-terminal end of the largest subunit of eukaryotic RNAPn (Table 28.1). This sequence is readily accessible on the surface of the large protein complex and is commonly referred to as the C-terminal structural domain (CTD) of RNAPII. mAb8WG16 has been used to purify the thymus of calves (Thompsonetal., 1990), yeast (EdwardsetaL, 1990), and human cells (Maldives, 1990). mAb8WG16 has been used to purify the thymus of calves (Thompsonetal., 1990), yeast (EdwardsetaL, 1990), and other proteins. ), yeast (EdwardsetaL,1990) and RNAPII in human cells (Maldonadoetal.,1996). In fact, the first application of PR-mAb purified yeast RNAPII was in RNAPII crystallization studies (Crameretal., 2000). We have described the detailed procedure for purifying RNAPU using PR-mAb (ThompsonandBurgess,1996). In most cases, purification of RNAPII from feedstocks requires a direct mixing purification procedure prior to the use of immunoaffinity resins. mAb8RBI3 is a highly cross-reactive PR-mAb that has been shown to be very effective in purifying bacterial nuclear RNA polymerase (coreRNAP) (Bergendahletal., 2003; Probascoetal., 2007). This PR-mAb reacts with the highly conserved "news-flap" structural domain of most RNA polymerases of bacterial origin (E.S. Stalder, unpublished). It is a major binding site for the sigma subunit (Kuznedelovetal., 2002), so RNA polymerase can be purified on 8RB13-agarose columns (lacking a sigma subunit). Figure 28.4 Lane 3 shows the nuclear RNA polymerase purified from E. coli. Given the cross-reactivity of this mAb, it is also possible to purify nuclear RNA polymerase from Bacillus subtilis (lane 4). Figure 28.4 also shows the RNA polymerase purified from E. coli using mAbNT73 (lane 2), which is a mixture of holoenzyme and coreenzyme, the two enzyme fractions of which can be separated by ion-exchange chromatography (Thompsonetal., 1992). The peptide corresponding to the major sigma factor is missing in the purification of the nuclear RNA polymerase from E. coli and B. subtilis sources. We believe that 5% to 10% of mAb isolated by standard hybridoma techniques may be valid PR-mAb, but even so, another approach can be used, i.e., epitope labeling of proteins with epitopes of mAb that have been identified as PR-mAb. The method of purifying the tag is described in Chapter I6. Therefore, we will cover this only briefly and only in relation to PR-mAb and its antigenic epitopes. The ability to label proteins with epitopes and to purify them by multilight group reactions with PR-mAb is a property of the antibody itself, rather than a characteristic of the reaction conditions in the presence of epitopes. We have developed epitope-tagging systems for three PR-mAb, NT73, IIB8, and 8RB13 (DuellmanetaL, 2004; Thompsonetal.,2003; E.S. Stalder, unpublished data). We call these epitopes "soft labels (3 this-ags)" (BurgessandThompson, 2 00 2). These epitopes are listed in Table 28.1, and these tags have been granted patents (Burgessetal., 2007). All of our PR-mAb isolations have used full-length proteins as immunogens; therefore, localization of such mAb epitopes is a laborious process. The use of synthetic peptides as immunogens for PR-mAb isolation, although feasible, has not been used by us. PR-mAbIIB8PR-which reacts with human TFIffi-was localized by targeted mutagenesis using phage display technology (Duellmanetal., 20 0 4). In contrast, the two PR-mAb (rnAbNT73 and mAb8RBI3) that react with the maximal subunit of E. coli RNA polymerase were first localized non-strictly by the ordered fragmentladder method (orderedfragmentladdermeth-o d) (BurgessetaL, 2000; Ra o etal., 1996), and then pinpointed by fine deletion analysis and oligonucleotide tagging of unrelated proteins (Thompsonetal., 2003; E.S. Stalder, unpublished data) to which we have antibodies. This oligonucleotide tag is constructed by fusing an oligonucleotide containing a sequence encoding an epitope compatible with the target protein reading frame to the target protein gene. In proof-of-principle studies of this technique, we used green photoprotein (GFP) as the target protein (DuellmanetaL, 2004; Thompsonetal., 2003). mAbNT73 epitope tags can be fused to either the N- or C-terminal end of GFP but, for other target proteins, may be fused to the N-terminal or C-terminal end of GFP. may depend on the accessibility of its end. For o.. , both ends are readily accessible in its crystal structure (Ding et al. 11, 1998).111 The epitope of octa-dagger 81^13 has been used as a tag in E. coli and mammalian cell culture sys For more product details, please visit Aladdin Scientific website.
