Optimizing Soluble Expression of Organophosphorus Acid Anhyrdolase (OPAA) Zingeber Udani BIOL 493 Fall 2007 1
Abstract Organophosphorus acid anhydrolase (OPAA) is a proteolytic enzyme expressed by many plant and bacterial species; several examples have demonstrated significant fortuitous hydrolytic activity against organophosphate pesticides and chemical warfare agents. While the opaa gene from Stenotrophomonas maltophilus has been previously expressed in Escherichia coli, the protein product tended to form insoluble, inactive, protein aggregates. Insoluble protein formation typically indicates improper folding of polypeptide chains and is due to inappropriate associations, especially between hydrophobic patches, during periods of rapid protein expression. Soluble protein is required for enzymatic activity. High levels of soluble protein are required to meet the demand for potential commercial pesticide detoxification applications, and are needed for the further characterization of the S. maltophilus OPAA, including structural analysis via x-ray crystallography. In the current study, expression of an intein-tagged OPAA was compared in Escherichia coli JM109, a general laboratory cloning strain, and in E. coli C43 (DE3), a strain optimized for the expression of soluble recombinant proteins. In addition, the effects of arginine and alanine supplementation on OPAA solubility during cell growth were observed. Expression of soluble OPAA, as gauged by SDS-PAGE and by enzymatic activity assay, was found to be optimized when expression was carried out in E. coli JM109 using growth media supplemented with 20 mm arginine. Introduction: Organophosphorus pesticides and chemical warfare agents can bind covalently within the active site of acetylcholinesterase (AChe), thereby blocking the nerve impulses in the peripheral nervous system, leading to paralysis and death (Quiocho and Nickitenko 2004). Organophosphorus acid anhydrolase (OPAA), an enzyme naturally found in many plant and bacterial species, belongs to the expanding class of known enzymes capable of detoxifying organophosphorus compounds (Hill et al. 2001). The OPAA of Alteromonas sp. JD6.5 has been well-characterized against G-type nerve agents, including soman and sarin (Cheng et al. 1999); however, a structural model of this protein is not currently available. Improvements in the catalytic activity of OPAA against chemical warfare agents may be possible through the site-directed modification of its active site; however, the lack of a structural model for this protein impedes this process (Kim and Lee 2001). 2
One factor that has affected the ability of this enzyme to be crystallized is the difficulty of purifying the protein in large quantities due to its tendency to form insoluble inclusion bodies when over-expressed in Escherichia coli. Numerous techniques have been developed that have lead to the successful expression of soluble recombinant proteins in other expression systems. The use of specially developed bacterial strains, altered growth conditions, and growth media supplements has been reported to be successful with other challenging proteins when overexpressed in E. coli. Two specific E. coli strains, C41(DE3) and C43(DE3), have proven to be especially advantageous in the soluble expression of difficult recombinant proteins (Sorensen and Mortensen 2005). The addition of arginine to growth media during protein expression appears to suppress aggregation of proteins during refolding (Arakawa and Tsumoto 2003). Similar studies suggest that proline, a natural osmolyte proline, may likewise result in the destabilization of protein aggregates and promote successful expression of active recombinant proteins (Ignatova and Gierasch 2006). The purpose of the current study is to compare the expression of the intein-tagged opaa gene from S. maltophilus in E. coli JM109 and in E. coli C41 (DE3) while observing the coupled effects of media supplementation with proline and arginine. Materials and Methods: Bacterial Culturing. Escherichia coli XL10 (Stratagene, Inc., La Jolla CA) possessing the plasmid ptwin/opaa SM (Swenson 2005), which drives expression of the opaa gene from Stenotrophomonas maltophilus as a fusion with the Mxe GyrA intein and chitin-binding 3
domain of ptwin, was cultured in LB media containing 150 µg/ml ampicillin at 37 C for routine maintenance. For protein expression, the plasmid was introduced into competent E. coli BL21 (Novagen, San Diego, CA) or E. coli C43(DE3) (Lucigen, Middleton, WI) by electroporation (Bio-Rad Laboratoties, Hercules, CA). Transformants were selected on LB plates (E. coli BL21) or YT plates (C43(DE3)) containing 150 µg/ml ampicillin. Protein Expression A single colony isolate of E. coli BL21 or E. coli C43(DE3) possessing ptwin/opaa SM was used to inoculate 5 ml LB containing 150 µg/ml ampicillin; this culture was incubated for 16 hours and used to inoculate LB broth, with and without supplementation with 20mM Proline or Arginine, at a 1% inoculum rate. Cultures were incubated at 16 C to an optical density (A625nm) of 0.5 to 0.6. Protein expression was induced by the addition of isopropyl β-d-1 thiogalactopyranoside (IPTG) to a final concentration of 1 mm, and incubation was continued for 18 hours at 16 C. Following induction, cells were harvested and pelleted by centrifugation (10 000 x g for 10 minutes at 4 C) and frozen at -20 C. For protein isolation, the bacterial cell pellet was resuspended in 20mM Tris-HCL, ph 8.6, at the rate of 4 ml buffer/g cells and lysed by sonication (1 minute sustained delivery at 80% load) using a flat tip with ½ in tapped horn on a 150V/T Ultrasonic Homogenizer (Biologics Inc. Manassas, Virginia) Sonication was repeated for a total of three cycles with 10 minute incubation on ice between each cycle. Cell debris was pelleted by centrifugation (10 000 x g for 30 min.) at 4 C and the soluble protein was 4
concentrated by adding ammonium sulfate to the supernatant at the rate of 0.472 g ammonium sulfate/ml and centrifuging as before to pellet the protein. SDS-PAGE SDS-PAGE was used to test the presence of the OPAA/intein fusion protein in both the soluble and insoluble cellular fractions. Protein samples were run at 180 V (< 50 ma) in a 1X Tris-Glycine-SDS running buffer. Incubation was done at room temperature for 2-16 hours for both the staining and destaining procedures. DFP Assay Cell-free extracts (CFE) were assayed for OPAA activity by monitoring the hydrolysis of fluorine from diisopropylfluorophosphate (DFP) using a Fluoride-specific ion-selective probe with the 5-Star Orion Portable Multimeter (Thermo Electron Corporation, Waltham, MA). The reaction was initiated by adding CFE to a solution containing 1 mm DFP in 50 mm NH 4 Cl, ph 8.5 and monitoring the change in fluoride concentration (µm) at five-second intervals for 10 minutes. Initial velocities were determined and used to compare relative activities. Protein Concentration Determinations. Protein concentrations were determined using a Lambda 25 UV/VIS spectrometer (PerkinElmer, Waltham, Massachusetts) at 260nm and 280nm. All samples were diluted to same concentrations for use in proceeding procedures. 5
Intein cleavage Intein Cleavage was achieved by the addition of Dithiothreitol (DTT) and incubation at room temperature overnight. Data analysis Data obtained from the DFP Assay was synthesized using Microsoft Excel. Results Protein Expression A 75 kda protein band indicative of OPAA/intein over-expression was observed only when LB media was supplemented with 20 mm arginine, both for E. coli BL21 and E. coli C43(DE3) (Figure 1). 1 2 3 4 5 6 7 8 9 Figure 1. SDS-PAGE for both strain in soluble fraction. Lane 1: E. coli C43(DE3) ptwin/opaa SM CFE grown with 20 mm arginine supplementation; Lane 2: E. coli C43(DE3) ptwin/opaa SM CFE grown with 20 mm proline supplementation; Lane 3: E. coli C43(DE3) ptwin/opaa SM CFE grown without supplementation; Lane 4: E. coli C43(DE3) ptwin; Lane 5: E. coli BL21 ptwin/opaa SM CFE grown with 20 mm arginine supplementation; Lane 6: E. coli BL21 ptwin/opaa SM CFE grown with 20 mm proline supplementation; Lane 7: E. coli BL21 ptwin/opaa SM CFE grown without supplementation; Lane 8: E. coli BL21 ptwin; Lane 9: Molecular weight standard. 6
DFP Assays Table 1. Shows the activity of OPAA against DFP before and after cleavage. Ave. Initial Samples Velocity um F/ 5s/ mg BL21 ptwin 0.1048 BL21 OPAA 0.3946 BL21 OPAA+Pro 0.6399 BL21 OPAA+Arg 0.7413 BL21 OPAA+Arg (Cleaved) 8.355 OMGC43(DE3) ptwin 0.5064 C43(DE3) OPAA 0.3582 C43(DE3) OPAA+Pro 0.395 C43(DE3) OPAA+Arg 0.21 C43(DE3) OPAA+Arg (Cleaved) 1.19 The cleaved protein showed activity when compared with the uncleaved proteins. Table 2. ANOVA test comparing between treatments, strains, and combinations of the two factors ANOVA Tests F F Crit. BL21 Between Treatments 17.425 3.49 C43(DE3) Between Treatments 0.474 3.49 BL21 Argenine vs. C43(DE3) Argenine 48.47 5.98 BL21 Arg. Vs. BL21 Pro 1.145 5.987 Testing between treatments for the E. coli BL21 showed statistical difference as evidenced by the higher F value than the F critical value. Treatments between the mutant strain E. coli C43(DE3) on the hand does not show significant statistical difference. Comparing the two different strains treated with the same LB media enhancement showed significant statistical difference. Using the same bacterial strain with arginine and proline LB media enhancement showed no significant statistical difference. 7
Discussion: Achieving sufficient quantity of OPAA using bacterial cell machinery and recombinant DNA has proven to be difficult. One factor contributing to this problem is the tendency of recombinant proteins to form inclusion bodies when over-expressed in E. coli thus; forming insoluble bodies (Swenson 2005).. These inclusion bodies are insoluble and does not have enzymatic activity, furthermore, it can be toxic to the host cell. These inclusion bodies are formed because the folding conditions of over-expressed proteins are different than in native organisms. In addition, high levels of expression favor improper associations Supplementation of LB media with arginine permitted in the over-expression of the OPAA/intein fusion protein in a soluble form. This can be gauged by the SDS-page result. DFP Assay and the ANOVA tests showed that arginine is the favored treatment and BL21 is the favored bacterial strain for this process. Previous studies have suggested that arginine binds to polypeptide chains during protein folding and may help solubilize the unfolded state allowing the protein more time to find its most energetically stable folded form and that this may be a natural mechanism used by cells to suppress protein aggregation during times of thermal stress (Arakawa and Tsumoto 2003). The presence of a thick band in lane 1 and 5 of Figure 1 shows that there is an observed over expression of protein when LB media is treated with arginine. However; this treatment is only needed at the expression level rather than the folding level of proteins expressed. Further study on the development of proper refolding of aggregate proteins could yield to sufficient amounts of functional OPAA needed for the purification and crystallization process. 8
While the protein expressed was fused with an intein tag, there was no activity noted in the DFP Assay. However, enzymatic activity was observed after the intein tag was cleaved from the protein of interest. This suggests that intein tag can inhibit proper functioning of the OPAA enzyme. These results suggest that the high-level expression of soluble OPAA/intein fusion protein can be enhanced with the supplementation of bacterial medias with 20 mm arginine. The intein tag would permit the rapid purification of OPAA out of a cell-free extract in an active form that would be conducive to future studies with protein crystallization. Crystallization is required for the construction of an x-ray crystallographic model of the OPAA protein. The availability of an atomic structure of this protein is necessary for the rational modification of its active site toward improved detoxifying capability against organophosphorus pesticides and chemical warfare agents. 9