No protein is detected in bacterial sonicate ing junctions sh sequenced to Optimize culture conditions to improve yield.Investigat te the effect of cell strain mediun nditions.Exact conditions will .Analyse a small aliquot of an overnight culture by SDS-PAGE.Generally,a highly ein will be visible by Coo ssier blue staini ng when 5-10 ul of ar induced cultu hoseis-1.0 is loaded on the nd cells ed with the rental ormed host E. ould b 。】。 and r of the fusio resence of Check for expression by immunoblotting.some fusion proteins may be masked on an rylamide gel of induced cells and transfer the proteins to a nitrocellulose or PVDF membrane (such as HybondC or Hybond-P).Detect fusion protein using anti-GST or anti-His antibody. Most of fusion protein is in the post-sonicate pellet .Check cell disruption procedure.Cell disruption is seen by partial clearing of the suspension or by microscopic examination.Addition of lysozyme(0.1 volume of a 10 mg/ml lysozyme solution in 25 mM Tris-HCl,pH 8.0)prior to sonication may improve results.Avoid frothing as this may denature the fusion protein. Reduce sonication since over-sonication can lead to co-purification of host proteins with the fusion protein. Fusion protein may be produced as insoluble inclusion bodies.Try altering the growth conditions to slow the rate of translation,as suggested below.It may be necessary to combine these approaches.Exact conditions must be determined empirically for each fusion protein. Lower the growth temperature(within the range of +20 to +30C)to improve solubility. Decrease IPTG concentration to<0.1 mM to alter induction level. Alter time of induction. Induce for a shorter period of time. Induce at a higher cell density for a short period of time. 10
10 No protein is detected in bacterial sonicate • Check DNA sequences. It is essential that protein-coding DNA sequences are cloned in the proper translation frame in the vectors. Cloning junctions should be sequenced to verify that inserts are in-frame. • Optimize culture conditions to improve yield. Investigate the effect of cell strain, medium composition, incubation temperature and induction conditions. Exact conditions will vary for each fusion protein expressed. • Analyse a small aliquot of an overnight culture by SDS-PAGE. Generally, a highly expressed protein will be visible by Coomassie™ blue staining when 5–10 µl of an induced culture whose A600 is ~1.0 is loaded on the gel. Non-transformed host E. coli cells and cells transformed with the parental vector should be run in parallel as negative and positive controls, respectively. The presence of the fusion protein in this total cell preparation and its absence from a clarified sonicate may indicate the presence of inclusion bodies. • Check for expression by immunoblotting. Some fusion proteins may be masked on an SDS-polyacrylamide gel by a bacterial protein of approximately the same molecular weight. Immunoblotting can be used to identify fusion proteins in these cases. Run an SDS-polyacrylamide gel of induced cells and transfer the proteins to a nitrocellulose or PVDF membrane (such as Hybond™-C or Hybond-P). Detect fusion protein using anti-GST or anti-His antibody. Most of fusion protein is in the post-sonicate pellet • Check cell disruption procedure. Cell disruption is seen by partial clearing of the suspension or by microscopic examination. Addition of lysozyme (0.1 volume of a 10 mg/ml lysozyme solution in 25 mM Tris-HCl, pH 8.0) prior to sonication may improve results. Avoid frothing as this may denature the fusion protein. • Reduce sonication since over-sonication can lead to co-purification of host proteins with the fusion protein. • Fusion protein may be produced as insoluble inclusion bodies. Try altering the growth conditions to slow the rate of translation, as suggested below. It may be necessary to combine these approaches. Exact conditions must be determined empirically for each fusion protein. - Lower the growth temperature (within the range of +20 to +30 °C) to improve solubility. - Decrease IPTG concentration to < 0.1 mM to alter induction level. - Alter time of induction. - Induce for a shorter period of time. - Induce at a higher cell density for a short period of time. - Increase aeration. High oxygen transport can help prevent the formation of inclusion bodies
usion protein. .Alter extraction conditions to improve solubilization of inclusion bodies(see Chapter 5). Quantification of fusion proteins Fsio protnust be purified to homogncity and ufdstandard protein assay. The relative yield of fusio determined by measuring the absorbance a(ble for bo GSTand (io) The yield of protein may also be determined by standard chromogenic methods (gLowry,BCA,Bradfo d,etc.) .Immunoassays can be used for quantification if a suitable standard curve can be oduced.In this the fuc ocreeninghye answer is required rified standard is ailabl c is alsqua articularly as,for example,when testing fractions from a purification
11 It may be necessary to combine the above approaches. Exact conditions must be determined empirically for each fusion protein. • Alter extraction conditions to improve solubilization of inclusion bodies (see Chapter 5). Quantification of fusion proteins Fusion proteins must be purified to homogeneity and quantified using a standard protein assay. • The relative yield of fusion protein can often be determined by measuring the absorbance at 280 nm (suitable for both GST and (His)6 fusion proteins). • The yield of protein may also be determined by standard chromogenic methods (e.g. Lowry, BCA, Bradford, etc.). • Immunoassays can be used for quantification if a suitable standard curve can be produced. In this case, the fusion protein does not have to be purified for quantification as long as a purified standard is available. The immunoassay technique is also particularly suitable for screening large numbers of samples when a simple yes/no answer is required, as, for example, when testing fractions from a purification
CHAPTER 3 GST fusion proteins Amplification Glutath erase (GST)Gene Fusion System is an integrated range of products for detection of GS n proteins in E he charac- teristics of GS 7 and Figure 1 shows the structure of Glutathione tion steps Glutathione S-ransferase ned in pGEX-IN M.58500 0.43±0.07m tofocusing) 508±Q7m GST class hybrid of Alpha and Mu characteristics Table 7. General considerations for the amplification of fusion proteins are discussed in Chapter 2. In the GST gene fusion system expression is under control of the tac promoter,which is 尚iCre The host E.coli BL21 is a protease-deficient strain specifically selected to give a high level of expression of GST fusion proteins. edia pl Longterm storage Table 8. Use an alternative strain for cloning and maintenance of the vector (e.g.JM105)as BL21 does not transform well. 业 Using E.coli strains that are not protease-deficient may result in proteolysis of the fusion protein,seen as multiple bands on SDS-PAGE or Western blots. 13
13 CHAPTER 3 GST fusion proteins Amplification Glutathione S-transferase (GST) Gene Fusion System is an integrated range of products for the amplification, purification and detection of GST fusion proteins in E. coli. The characteristics of GST are shown in Table 7 and Figure 1 shows the structure of Glutathione Sepharose used in the purification steps. Glutathione S-transferase Naturally occurring Mr 26 000 protein Can be expressed in E. coli with full enzymatic activity Properties as determined in pGEX-1N Dimer Molecular Weight Mr 58 500 Km (glutathione) 0.43 ± 0.07 mM Km (CDNB) 2.68 ± 0.77 mM pI (chromatofocusing) 5.0 GST class hybrid of Alpha and Mu characteristics Table 7. Genotype F- , ompT, hsdS (rB - , mB - ), gal (52, 53) Growth conditions Resuspend lyophilized cultures in 1 ml of L-broth. Grow overnight before plating onto L-broth media plates Long term storage Mix equal volumes of stationary phase culture (grown in L-broth) and glycerol. Store at -70 °C. Revive frozen glycerol stocks by streaking onto L-broth media plates Use an alternative strain for cloning and maintenance of the vector (e.g. JM105) as BL21 does not transform well. Using E. coli strains that are not protease-deficient may result in proteolysis of the fusion protein, seen as multiple bands on SDS-PAGE or Western blots. CH2 C CH2 H OH S C O H N C O O N H O C C O NH3+ O O Fig. 1. Glutathione is attached to Sepharose by coupling to the oxirane group using epoxy-activation. The structure of glutathione is complementary to the binding site of the glutathione S-transferase binding site. General considerations for the amplification of fusion proteins are discussed in Chapter 2. In the GST gene fusion system expression is under control of the tac promoter, which is induced using the lactose analogue isopropyl b-D-thiogalactoside (IPTG). Induced cultures should be left to express GST fusion proteins for several hours before the cells are harvested. The host E. coli BL21 is a protease-deficient strain specifically selected to give a high level of expression of GST fusion proteins. Table 8
The vectors GEX vectors (pGEX-T.pGEX-B pGEX-,pGEX-2TK)are three reading ith n ea vector are shown pGEX-6P-1.pGEX-6P-2.pGEX-6P-3 PreScission Protease TromnAMPdependentpro kinase Table9. ease vectors GST offer the mos effi hod for e vage and on prote ultancous protease or the The prot a low e pert ivage enzyme a nd GST tagare Purification For simple,one step purification of GST fusion proteins,several products have been designed to meet specific purification needs,as shown in Table 10. lwegrepacked Comment roteinoroa GST MicroSpin Purification Module GSTan EE 1ml 10-12mg GSTrap FF5 ml 50-60mg Prepacked column.ready to use Glutathione Sepharose4B 8mg per ml For packine small columns and other formats Glutathione Sepharose 4 Fast Flow 10-12 mg per ml Table 10.Summary of purification options for GST fusion proteins. Characteristics of GSTrap FF and Glutathione Sepharose are given in Appendix 5. Re-use of purification columns depends upon the nature of the sample and should only be performed with identical samples to prevent cross contamination. Batch p aratio are fre ned in the literature.Hov er the packed colu mns and easily packed high flow rate Glutathione Sepharose venient alter natives.Batch essible or when the occasionally used if it orein in the lysate is at ve ry low entrations (hoth could a eyiedistodceahaPpurntcatiom ar to give a low yield fr the firs A more conven ient alternativ to im sample through the column several times
14 Table 9. Column (prepacked) Amount of GST Comment or Media** fusion protein for a single purification GST MicroSpin Purification Module Up to 400 µg Ready to use, prepacked columns, buffers and chemicals High throughput when used with MicroPlex™ 24 Vacuum (up to 48 samples simultaneously) GSTrap FF 1 ml 10–12 mg Prepacked column, ready to use GSTrap FF 5 ml 50–60 mg Prepacked column, ready to use Glutathione Sepharose 4B 8 mg per ml For packing small columns and other formats Glutathione Sepharose 4 Fast Flow 10–12 mg per ml For packing high performance columns for use with purification systems and scaling up Re-use of purification columns depends upon the nature of the sample and should only be performed with identical samples to prevent cross contamination. Batch preparation procedures are frequently mentioned in the literature. However the availability of prepacked columns and easily packed high flow rate Glutathione Sepharose provide faster, more convenient alternatives. Batch preparations are occasionally used if it appears that the tag is not fully accessible or when the protein in the lysate is at very low concentrations (both could appear to give a low yield from the first purification step). A more convenient alternative to improve yield is to decrease the flow rate or pass the sample through the column several times. pGEX-6P-1, pGEX-6P-2, pGEX-6P-3 PreScission Protease pGEX-4T-1, pGEX-4T-2, pGEX-4T-3 Thrombin pGEX-5X-1, pGEX-5X-2, pGEX-5X-3 Factor Xa PGEX-2TK Thrombin, c-AMP dependent protein kinase Allows detection of expressed proteins by direct labelling in vitro pGEX6P PreScission Protease vectors offer the most efficient method for cleavage and purification of GST fusion proteins. Site specific cleavage is performed with simultaneous immobilization of the protease on the column. The protease has a high activity at a low temperature so that all steps can be performed in the cold room to protect protein integrity. Cleavage enzyme and GST tag are removed in a single step. Purification For simple, one step purification of GST fusion proteins, several products have been designed to meet specific purification needs, as shown in Table 10. Table 10. Summary of purification options for GST fusion proteins. **Characteristics of GSTrap FF and Glutathione Sepharose are given in Appendix 5. The vectors pGEX vectors (pGEX-T, pGEX-P, pGEX-X, pGEX-2TK) are available in all three reading frames with a range of cleavage recognition sites as shown in Table 9. The same multiple cloning sites in each vector ensure easy transfer of inserts. The vectors carry the lacIq gene, so there are no specific host requirements for expression of fusion proteins. Vector control regions and the reading frame of the multiple cloning site for each pGEX vector are shown in Appendix 1
ment should also be made a cording to the r nceds of the urificat P uide to aid in the nd key po onsidcerarehigehlightcdhere ection of the correct For a single purification of a small quantity of product or for high throughput screening MicroSpin columns using centrifugation or MicroPlex 24 Vacuum are convenient and simple to use. the ideal solution and can be used with a syringe,a peristaltic pump or a chromatography system. Toincrease capacity use several GSTrap FF() en larger capacit eq hione Sepharo rast riow a suitable column(details of coumn packing procedures are outined in ppendix inating manual errors .For laborat nts in which all experimental data must be recorded and and cont sign chromatography system is recommended.mu elutio n steps that t can only be performed by a chromatography system. GST MicroSpin Purification Module The GST MicroSpin Purification Module is useful for large numbers of lysates and for checking samples during the optimization of amplification for 50 purifications. 10X PBS 1.4 M NaCl,27 mM KCI,101 mM Na,HPO 18 mM KH2PO pH 7.3 Reduced glutathione:0.154 g Dilution buffer: 50 mM Tris-HCI.pH 8.0 。IPTG: 500mg .MicroSpin columns:50 units Reagents are prepared as follows: 1X PBS: Dilute 10X PBS with sterile water.Store at +4 C. Glutathoneeli buffer:Pour theenireofiibufferint the bottentning the IPTG 100 mM:
15 Monitor purification steps by using one or more of the detection methods referred to in this handbook. The choice of purification equipment should also be made according to the needs of the purification. Appendix 8 provides a guide to aid in the selection of the correct purification solution and key points to consider are highlighted here. • For a single purification of a small quantity of product or for high throughput screening MicroSpin columns using centrifugation or MicroPlex 24 Vacuum are convenient and simple to use. • For purification of larger quantities of fusion proteins GSTrap FF columns provide the ideal solution and can be used with a syringe, a peristaltic pump or a chromatography system. • To increase capacity use several GSTrap FF columns (1 ml or 5 ml) in series or, for even larger capacity requirements, pack Glutathione Sepharose 4 Fast Flow into a suitable column (details of column packing procedures are outlined in Appendix 6). • For simple and reproducible purification a chromatography system such as ÄKTA™prime is a significant advantage, recording the purification process and eliminating manual errors. • For laboratory environments in which all experimental data must be recorded and traceable, where method development, optimization and scale up are needed, a computer controlled ÄKTAdesign chromatography system is recommended. • Experiments such as protein refolding or method optimization require linear gradient elution steps that can only be performed by a chromatography system. GST MicroSpin Purification Module The GST MicroSpin Purification Module is useful for screening small or large numbers of lysates and for checking samples during the optimization of amplification or purification conditions. Each module contains reagents sufficient for 50 purifications. • 10X PBS: 1.4 M NaCl, 27 mM KCl, 101 mM Na2HPO4, 18 mM KH2PO4, pH 7.3 • Reduced glutathione: 0.154 g • Dilution buffer: 50 mM Tris-HCl, pH 8.0 • IPTG: 500 mg • MicroSpin columns: 50 units Reagents are prepared as follows: 1X PBS: Dilute 10X PBS with sterile water. Store at +4 °C. Glutathione elution buffer: Pour the entire contents of dilution buffer into the bottle containing the reduced glutathione. Shake until completely dissolved. Store as 1–20 ml aliquots at -20 °C. IPTG 100 mM: Dissolve contents of the IPTG vial in 20 ml sterile water. Store as 1 ml aliquots at -20 °C