UNIT 6.3 Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies (UNITS 5.1 & 6.1). Inclusion bodies are normally formed in the cytoplasm alternatively, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies recovered from cell lysates by low-speed centrifugation are heavily contaminated with E. coli cell wall and outer membrane components. The latter are largely removed by selective extraction with detergents and low concentrations of either urea or guanidine���HCl to produce so-called washed pellets. These basic steps result in a signifi- cant purification of the recombinant protein, which usually makes up ���60% of the washed pellet protein. The challenge, therefore, is not to purify the recombinant-derived protein, but to solubilize it and then fold it into native and biologically active protein. Basic Protocol 1 describes preparation of washed pellets and solubilization of the protein using guanidine���HCl. The extracted protein, which is unfolded, is either directly folded as described in UNIT 6.5 or further purified by gel filtration in the presence of guanidine���HCl as in Basic Protocol 2. A support protocol describes the removal of guanidine���HCl from column fractions so they can be monitored by SDS-PAGE (UNIT 10.1). BASIC PROTOCOL 1 PREPARATION AND EXTRACTION OF INSOLUBLE (INCLUSION-BODY) PROTEINS FROM ESCHERICHIA COLI Bacterial cells are lysed using a French press, and inclusion bodies in the cell lysate are pelleted by low-speed centrifugation. The pellet fraction is washed (preextracted) with urea and Triton X-100 to remove E. coli membrane and cell wall material. Guanidine���HCl (8 M) and dithiothreitol (DTT) are used to solubilize the washed pellet protein. Extraction with the denaturant simultaneously dissociates protein-protein interactions and unfolds the protein. As a result, the extracted protein consists (ideally) of unfolded monomers, with sulfhydryl groups (if present) in the reduced state. Materials E. coli cells from fermentation (UNIT 5.3) containing the protein of interest Lysis buffer (see recipe) Wash buffer (see recipe), with and without urea and Triton X-100 Extraction buffer (see recipe) 250- and 500-ml stainless steel beakers 0.22-��m syringe filters (e.g., Millex from Millipore) 20-ml disposable syringe Additional equipment for breaking cells, homogenizing cells and pellets and centrifuging at low and high speeds (UNIT 6.2) Break cells and prepare clarified lysate 1. Place thawed E. coli cells in a stainless steel beaker. Add 4 ml lysis buffer per gram wet weight of cells. Keep bacterial cells cool by placing the beaker on ice in an ice bucket. The cells can be pretreated with lysozyme prior to lysis in the French press. Lysozyme treatment involves incubating cells ���20 min at 20�� to 25��C in lysis buffer supplemented Contributed by Ira Palmer and Paul T. Wingfield Current Protocols in Protein Science (1995) 6.3.1-6.3.15 Copyright �� 2000 by John Wiley & Sons, Inc. CPPS 6.3.1 Purification of Recombinant Proteins

with 200 ��g/ml lysozyme, with intermittent homogenization using a tissue grinder. It should be emphasized that this optional step is carried out before French press breakage and is not simply an alternative method of cell breakage (compare the comments made in the annotation to step 4 of UNIT 6.2). Its purpose is to aid removal of the peptidoglycan and outer membrane protein contaminants during the washing steps (steps 6 to 9 for further details see UNIT 6.1 and Fig. 6.1.5). An example of this approach is given in Basic Protocol 1 of UNIT 6.5. 2. Suspend cells using a Waring blender and homogenize using the Polytron tissue- grinder homogenizer until all clumps are disrupted, as described in UNIT 6.2, step 3. 3. Lyse cells with two passes through the French pressure cell operated at 16,000 to 18,000 lb/in2 (with the high-ratio setting, pressure gauge readings between 1011 and 1135), chilling the cell suspension to 4��C after each pass, as described in UNIT 6.2, steps 2 and 4. 4. Reduce the viscosity of the suspension by sonicating 5 min at full power with 50% duty cycle (on for 5 sec, off for 5 sec) using an ultrasonic homogenizer, as described in UNIT 6.2, step 5. 5. Clarify the lysed cell suspension by centrifuging 1 hr at 22,000 �� g (12,000 rpm in a JA-14 rotor in a Beckman J2-21M centrifuge), 4��C. Unbroken cells, large cellular debris, and the inclusion body protein will be pelleted. The JA-14 rotor uses 250-ml centrifuge bottles. For processing smaller volumes the Beckman JA-20 rotor (or equivalent) with 50-ml tubes can be used, at 13,500 rpm (22,000 �� g). The procedure for dealing with insoluble inclusion-body proteins now diverges from that for purifying soluble proteins (UNIT 6.2). Prepare washed pellets 6. Carefully pour off the supernatant from the pellet. Using a tissue homogenizer, suspend the pellet with 4 to 6 ml wash buffer per gram wet weight cells. Complete homogenization of the pellet is important to wash out soluble proteins and cellular components. Removal of cell wall and outer membrane material can be improved by increasing the amount of wash solution to 10 ml per gram cells. The concentration of urea and Triton X-100 in the wash buffer can be varied. The urea concentration is usually between 1 and 4 M higher concentrations may result in partial solubilization of the recombinant proteins. The usual detergent concentration is 0.5% to 5%. Triton X-100 will not solubilize inclusion body proteins it is included to help extract lipid and membrane-associated proteins. 7. Centrifuge the suspension 30 min at 22,000 �� g (12,000 rpm in JA-14), 4��C. Discard supernatant and, using the tissue homogenizer, suspend the pellet in 4 to 6 ml wash buffer per gram wet weight of cells. 8. Repeat step 7 two more times. If the supernatant is still cloudy or colored, continue washing the pellet until the super- natant is clear. 9. Suspend the pellet with wash buffer minus the Triton X-100 and urea, using 4 to 6 ml buffer per gram wet cells. Centrifuge 30 min at 22,000 �� g (12,000 rpm in JA-14), 4��C. The final wash removes excess Triton X-100 from the pellet. Current Protocols in Protein Science 6.3.2 Preparation and Extraction of Inclusion Bodies

If necessary the washed pellets can be stored at ���80��C. It is better to store material at this stage rather than after the extraction stage (see comments to step 13). Extract recombinant protein from washed pellets with guanidine���HCl 10. Using the tissue homogenizer, suspend the pellet with guanidine���HCl-containing extraction buffer. Use 0.5 to 1.0 ml buffer per gram wet weight of original cells if the extract will be subjected to gel filtration, and 2 to 4 ml buffer if the extract will be used in protein folding procedures. Perform this step at room temperature. To estimate the amount of recombinant protein in the washed pellets, use the following guidelines. (1) An expression level of 1% corresponds to ���1 mg recombinant protein per 1 g wet cells. (2) The recovery of highly aggregated recombinant protein in the washed pellets is ���75% that originally present in the cells. (3) About 60% of the total washed pellet protein is recombinant-derived. Thus, if 50 g cells is processed and the expression level is 5%, the washed pellets contain ���200 mg recombinant protein. The total amount of recombinant-derived protein in washed pellets can be directly deter- mined by measuring the total protein concentration or by analyzing the washed pellets via SDS-PAGE (see Support Protocol and UNIT 10.1) to determine the proportions of the protein constituents. For gel-filtration purposes, the pellets from 50 g wet weight E. coli cells are solubilized with 40 to 50 ml extraction buffer (see Basic Protocol 2) the concentration of recombinant protein in the extract will be 4 to 5 mg/ml. For direct protein folding (UNIT 6.5), the pellets are extracted with 100 to 200 ml buffer, and the concentration of recombinant protein 1 to 2 mg/ml. If the washed pellet is heavily contaminated with outer cell wall and peptidoglycan material, the extract must be diluted further with extraction buffer (usually 1:1 to 1:3) to reduce the viscosity before it can be used for chromatography. 11. Centrifuge the suspension 1 hr at 100,000 �� g (30,000 rpm in Ti45 rotor in a Beckman Optima XL-90 ultracentrifuge), 4��C. For volumes 250 ml the Beckman 70Ti rotor (capacity 6 �� 39 ml) can be used at 32,000 rpm (���100,000 �� g). 12. Carefully pour off the supernatant from the pellet. Filter the supernatant through a 0.22-��m syringe filter attached to a 20-ml disposable syringe. The filter removes unpelleted large cell wall debris that will clog most chromatography columns. 13. Use the clarified inclusion body extract for preparing folded protein (UNIT6.5) or purify further by gel filtration (see Basic Protocol 2). The extract can be stored at ���80��C until required. Freeze in plastic (or polyethylene) containers rather than glass. Divide sample into 10- to 20-ml aliquots instead of freezing in one large lot and fill containers to only 50% to 75% capacity. BASIC PROTOCOL 2 MEDIUM-PRESSURE GEL-FILTRATION CHROMATOGRAPHY IN THE PRESENCE OF GUANIDINE HYDROCHLORIDE Washed, extracted pellets (see Basic Protocol 1) contain 50% recombinant protein and are used as the starting material for purification of the protein of interest by gel-filtration chromatography. Superdex 200 gel-filtration medium, which allows high flow rates, is washed and packed into a column. The column is equilibrated at 4��C and the sample is applied. Assay of column fractions by gel electrophoresis in the presence of SDS is complicated by the fact that guanidine���HCl forms a precipitate with SDS. Therefore, preparing samples for gel analysis involves selective precipitation of protein from guanidine���HCl prior to Current Protocols in Protein Science 6.3.3 Purification of Recombinant Proteins