FIELD OF THE INVENTION
The present invention relates to the production of recombinant proteins in Dictyostelium discoideum. More particularly, the invention relates to the expression of hPDEs in Dictyostelium discoideum. In particular, the invention relates to the process of expressing hPDE4B2 and hPDE7A. Also, the present invention relates to substantially pure recombinant protein.
BACKGROUND OF THE INVENTION
Phosphodiesterase have been implicated in variety of diseases such as asthma, COPD (chronic obstruction of pulmonary disease), neurodegenerative diseases, depression, learning disorders, erectile dysfunction, memory functions and myocardial infarction. Till date, eleven members of PDE superfamily (PDE-11) have been identified based on their biochemical properties, expression, regulation and inhibitor selectivity. PDEs regulate intracellular concentration of key second messenger's cAMP and/or cGMP by hydrolyzing them to 5'AMP and/or 5'GMP. Their crucial role in cell signaling has designated them as attractive targets for drug development. Importantly, cAMP- specific PDE4 is viewed as an effective therapeutic target in number of inflammatory diseases including asthma and COPD. Several PDE4 inhibitors have been investigated but the occurrence of side effects such as nausea, emesis and headache has led to the search for new inhibitors with better efficacy profiles. Therefore, many leading pharmaceutical companies are exploring new therapeutic agents based on selective and potent inhibition of PDE4 subtype-specific isoform.
The PDE4 family has four subtypes (A, B, C and D) and each subtype has multiple splice variants with unique N-terminal region. Among the various subtypes of PDE4, it has been shown that PDE4D subtype is mainly responsible for the side effects associated with the PDE4 inhibitors. Moreover, PDE4D3 inhibition is also associated with heart failure and lethal cardiac arrhythmias. On the contrary, PDE4B plays a central role in immune cell function and T cell regulation (Manning et al., 1999; Arp et al., 2003) and thus play an important role in inflammation. In addition, the studies in PDE4B knock out mice indicate that PDE4B is essential for LPS-activated TNF-a response as opposed to PDE4D (Jin and Conti, 2002). There are four known splice variants of PDE4B subtype, PDE4B1, 4B2, 4B3 and 4B4 (Bolger, et al., 1993; McLaughlin et al., 1993; Huston et al, 1997; Shepherd et al, 2003). PDE4B2 is the predominant subtype expressed in neutrophils, monocytes and leukocytes suggesting that PDE4B2 is relatively specific target for discovery of anti-inflammatory drugs (Wang et al., 1999; Torphy, 1998). Thus, development of PDE4B2 inhibitor would offer greater therapeutic advantage over other PDE4 subtypes (Robichaud et al., 2002, Lehnart et al. 2005, Dastidar et al., 2007).

It is known that activation of CD4+T cells requires stimulation of the CD3 receptor as well as costimulation of another receptor. Costimulation of the CD28 receptor leads to full activation of CD4+T cells. It has been shown that PDE7A is up regulated in CD4+T cells after CDS and CD28 stimulation and therefore inhibition of PDE7A upregulation with an antisense oligo leads to inhibition of proliferation.
Natural sources for proteins, for example, as pharmaceutical, are often limited, very expensive to purify or simply not available. Because of the hazard of toxic or especially infectious contaminants, they can also be very problematical. Biotechnology and genetic engineering, on the other hand, now make possible economical and safe production of an entire series of proteins in sufficient amount by heterologous expression for a wide variety of application for example antibodies, hormones, interleukins, etc.
Human proteins for medical application must be identical to the natural protein in biochemical, biophysical and functional properties. Various expression systems have been explored for the production of recombinant PDE4 needed for high-throughput in vitro screening of new chemical entities. Recombinant human PDE4 have expressed in bacteria, yeast, baculovirus and mammalian expression systems. However, PDE4 expressed in E. coli have either low specific activity (Kovala et al., 1997) or accumulated in inclusion bodies necessitating proper refolding of the protein for its bioactivity (Richer et al., 2000). Yeast expression system has been explored mainly for analysis of drug-resistant mutants of mammalian PDEs (Pillai et al., 1993; Atienza and Colicelli, 1998). Both baculovirus and mammalian cells express recombinant PDE4 with comparable biological activities to that of native proteins (Saldou et al., 1998). This indicates that these host cells contain all the enzymes to perform post-translational modifications such as phosphorylation, necessary to achieve functional bioactive protein. Unfortunately, low level of expression obtained in stably transfected mammalian cells limits their use for purification of recombinant PDE4. Moreover, expression of PDEs in these stable clones decreases with time and passage (Salanova et al., 1998). Most of the recombinant PDEs are currently in Sf9 insect cells that offer very efficient method to produce bulk quantities of active PDE4. However, the disadvantage of baculovirus system is that the insect cells grow slowly and usually require expensive media. Thus, there is need in the art for the expression of PDEs in a more robust expression host that is fast, cost effective and produces large quantities of active PDE.
The single celled eukaryote Dictyostelium discoideum provides an attractive alternative for heterologous expression of recombinant human PDEs. Presently five different PDEs (Pdel, Pde2, Pde3, PdeD, PdeE) have been identified from Dictyostelium discoideum that play crucial role in regulating intracellular concentration of cAMP and cGMP second messengers similar to

higher eukaryotes (Saran et al., 2003). Among these Pde2 and Pde3 show high degree of amino acid sequence identity with mammalian catalytic domains of PDEs (Kuwayama et al., 2001; Shaulsky et al., 1998). In addition, Dictyostelium offer major advantage as an expression host because it can be grown and manipulated with the same ease as bacteria or yeast without compromising on the post-translational modification of the expressed mammalian proteins. The cellular slime mold of Dicytostelium discoideum is a free-living organism, easy to grow and maintain. Dicytostelium discoideum stains can grow on bacteria lawns with doubling time of about 3 hours, in bacterial suspensions to high densities (up to 1010 cells per liter) or in semi-synthetic media containing glucose, peptone and yeast extract where doubling time is about 12 hours. The life cycle of Dicytostelium discoideum consist of a growth and developmental phase. The developmental phase is triggered by starvation and is characterized by aggregation of previously single cells to form a muticellular organism which then differentiates to produce spores which can be stored over a prolonged period of time. Germination of spores in the presence of bacteria or rich media will allow renewed growth. During this developmental cycle, diffusible factors are produced and for at least one of them (cAMP) binding to its receptor induces transcription of a set of specific genes.
A number of heterologous proteins have been successfully expressed in Dictyostelium such as human gonadotropin and follicle stimulating hormone (Linskens et al., 1999), human choriogonadotropin (Heikoop et al., 1998), human muscarinic receptor M2 (Voith et al., 1995), human antithrombin III (Dingermann et al., 1991), green fluorescent protein (Gerisch et al., 1995) and soluble human Fas ligand (Lu et al., 2004).
SUMMARY OF THE INVENTION
In accordance with one aspect, there is provided an economical method for the production of recombinant PDEs.
In accordance with second aspect, there is provided method for the expression of PDEs in Dictyostelium discoideum.
In accordance with third aspects, there is provided a method for the expression of hPDE4B2 (Accession No. M97515) in Dictyostelium discoideum.
In accordance with fourth aspect, there is provided method for the expression of hPDE7A (Accession No. LI2052) in Dictyostelium discoideum.

In accordance with fifth aspect, there is provided substantially pure recombinant hPDE4B2 and hPDE7A determined from SDS-PAGE (Sodium dodecyl-polyacrylamide gel) and western blot analysis.
The term "Substantially pure" herein refers to the recombinant hPDE4B2 and hPDE7A having purity not less than 60%.
The details of one or more aspects of the invention are set froth in the description below. Other features, objects and advantages of the inventions will be apparent from the description.
DETAILED DESCRIPTION OF THE INVENTION
It has been found that the species of the Dictyostelium discoideum can be used as an efficient eukaryotic expression system for the production of recombinant protein.
In the present work, successful expression of recombinant PDEs in Dictyostelium discoideum is described for the first time. For this purpose, the method of producing a recombinant protein comprises steps:
a) Amplification of hPDEs using primers as follows: seq. ID 1 (5'
cgcggatccatgaaggagcacgggggc-3') and seq. ID 2 (5'-gcctcgagatgtatccacgggggacttg-3')
and cloning in pB17S vector;
b) transformation of host cell Dictyostelium discoideum with the cloned hPDEs;
c) culturing the host cell Dictyostelium discoideum expressing the desired gene in the
presence of antibiotic;
d) screening the expressed recombinant hPDEs by western blotting using specific antibody;
e) expansion of the positive clone expressing recombinant hPDEs in bulk;
f) preparation of cell lysates in stabilizing buffer;
g) purification of recombinant hPDEs by Ni-NTA His-Tag affinity chromatography;
h) testing the activity of purified recombinant hPDEs by cAMP assay using Discoverx kits.
Brief description of the drawings
Figure 1: Vector map of cloning site.
Figure 2: SDS-PAGE/Western blot analysis.
Figure 3: Immunofluorescence studies of expressed recombinant protein.
Figure 4: Elution peak distribution profile.
Figure 5: Michaelis-Menten kinetics of expressed recombinant hPDE4B2.

Examples
The examples mentioned below demonstrate the specific process for the expression of recombinant hPDEs. The examples are provided to illustrate the details of the invention and should not be constrained to limit the scope of the present invention.
Example 1: Amplification of hPDE4B2 using primers as follows; seq. ID 1 (5' cgcggatccatgaaggagcacgggggc-3,) and seq. ID 2 (5'-gcctcgagatgtatccacgggggacttg-3') and cloning in pB17S vector.
The full length hPDE4B2 cloned in mammalian expression vector pcDNAS.l (Bora et al., Biochem Biophys Res Commun. 2007 Apr 27; 356(1): 153-8) was used as template to reamplify the gene for cloning into Dictyostelium expression vector pB17S. The PCR was performed with Expand Long Template Polymerase (Roche) using the following set of primer sequences. Forward Primer: 5' cgcggatccatgaaggagcacgggggc-3' Reverse Primer: 5'-gcctcgagatgtatccacgggggacttg-3'
The PCR fragment was cloned in pB17S vector at BamHI and Xho I restriction sites in frame with N-terminal His-tag as shown in Fig. 1 (Muhia et. Al., J Biol. Chem. 2003 Jun 13;278(24):22014-22..

(Figure 1 Removed)
ATO CAT CAT CAT CAT CAT CAT CAT GAT GGT ACC GAG CTC GGA TCC ACT CGA G TAA TCT AGA
In this vector, hPDE4B2 is constitutively expressed under the control of Actin 15 promoter.
Example 2: Transformation of host cell Dictvostelium discoideum with the cloned hPDEs
The Dictyostelium axenic AX3 cells were transformed by electroporation method (Heikoop et al. 1998). Approx. 2 X 107 cells were washed twice with KK.2 buffer and centrifuged at 100 X g for
5 min. Then the cells were washed with electroporation buffer H-50 (20mM HEPES, 50mM KC1, lOmM NaCl, 1 mM MgSO4, 5mM NaHCO3, 1mM NaH2PO4, pH 7.0) and resuspended in 0.1 ml H-50 buffer. 10µg of recombinant pB17S-4B2 DNA was added to the cells. The cell suspension was transferred to cold 0.1 cm cuvette and electroplated in a BioRad Gene Pulsar at 0.85 kv, 25uF and 0.6 msec time constant. The cuvette was incubated on ice for 5 min and the cells were transferred to HL-5 medium in 10 cm2 tissue culture dishes.
Example 3; Culturing the host cell Dictyostelium discoideum expressing the desired gene in the presence of antibiotic
AX3 strain of Dictyostelium discoideum cells were grown axenically in HL-5 medium (14.3 g protease peptone, 7.15 g yeast extract, 6 g glucose, 0.626 g Na2HPO4 and 0.485 g KH2PO4 per liter, pH 6.5) at 22°C (Watts et al., Biochem J. 1970 Sep;l 19(2):171-4). Dictyostelium discoideum transformants were maintained at 22°C on HL-5 medium supplemented with 10 ug/ml aminoglycoside antibiotic G418. Since the plasmid contains G418 selection marker, only transformants survived after continuous G418 exposure. As the concentration of antibiotic G418 was increased from 10 u.g/ml, stable transformants formed colonies. For large-scale culture, log-phase AX3 cells (2 X 107 cells) were inoculated into 11 flask containing 500 ml HL-5 medium with 10 |ag/ml antibiotic G418. The flasks were incubated on a shaker at 180 rpm/22°C until the density reached log phase (4X106 cells/ml) for storage of spores, cells were harvest at a density of 2 X 107 cells/ml and washed with KK2 buffer (2.25 g KH2PO4 per liter, pH 6.2). The cells were resuspended in same buffer and spotted on non-nutrient agar plates. After 24h, spores were collected and stored in salt solution (0.6g NaCl, 0.5 g KC1 and 0.4 g CaCl2.2H2O) containing 80 % glycerol.
Example 4: Screening the expressed recombinant hPDEs by western blotting using specific antibody;
The transformants were subjected to antibiotic G418 selection up to 1 mg/ml. The stable transformants were picked and propagated in HL-5 medium containing 100µg/ml antibiotic G418.
For initial screening of recombinant positive clones, transformed cells grown in axenic medium were harvested at a cell density of 106 cells/ml. The cell extracts and the medium supernatant were separated by SDS-PAGE followed by western blot analysis. The expected band size (-92 Kd) of recombinant hPDE4B2 was detected by anti-PDE4B2 antibody in the cell extracts of clone number 7 and 11 (Fig. 2). The localization of expressed recombinant protein was done by
fluorescence studies in different developmental stages of Dictyostelium. As opposed to wild type cells, bright yellow fluorescence was observed in the spores of the fruiting body of hPDE4B2 transformed cells. No florescence was observed in the stalk or basal body of the fruiting body (Fig. 2C and D).

(Figure Removed)

However, the right size band was not observed in the media supernatant indicating that
recombinant hPDE4B2 was retained in the cytoplasm and not secreted into the medium.
The localization of expressed recombinant protein was done by fluorescence studies which are as follows.Immunofluorescence: AX3-4B2 Dictyostelium discoideum cells were grown in 4-well-Lab-Tek
chamber slides for 24 h at 37°C. The cells were fixed with 2% paraformaldehyde/0.1% Triton
X-100 for 20 min at room temperature. The cells were blocked in 10% FBS for 20 min at room
temperature. The cells were incubated with 1:100 dilution of appropriate primary antibody
followed by incubation with 1:500 diluted Alexa-conjugated secondary antibody. Cells were
analyzed under a fluorescent microscope TE-2000-E (Nikon Instech Co. Ltd., Japan)
Bright yellow fluorescence was observed in the transformed cells only as opposed to control wild type cells (Fig. 3).
(Figure 3 Removed)
Fig. 3: Immunoflorescence studies of expressed recombinant proteins
The recombinant protein was expressed in the cytoplasm. This further confirms that recombinant hPDE4B2 is expressed only in the cytoplasm of Dictyostelium discoideum.
Example 5: Expansion of the positive clone expressing recombinant hPDEs in bulk
In order to produce large quantities of hPDE4B2, two 500 ml flasks containing HL-5 medium with 100 (ag/ml geneticin were inoculated with 2 X 107 cells/ml AX3PDE4B2 transformed cells. The flasks were grown as shaken cultures at 22°C.
Example 6; Preparation of cell lysates in stabilizing buffer
The cells were, then, lysed in solubilizing buffer containing 45mM Tris-HCl, pH 7.5, 0.05% MgCl2, ImM EDTA, pH 8.0, 0.2 mM EGTA, 0.05 mM DTT, 0.01 % Triton X-100, 0.01 % glycerol and protease inhibitor cocktail followed by 5 cycles of free-thaw. The lysates were centrifuged at 3000 X g at 5 min. The lysates from stable transformants were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 8-10% gels and recombinant hPDE4B2 was detected by western blot (immounoblot). Cells harvested from 1 ml culture were lysed in Laemmli buffer and boiled for 3 min. The supernatant were subjected to SDS-PAGE followed by wet transfer of proteins to nitrocellulose membranes. Standard western
blot procedures were used to detect PDE4B2 protein with primary rabbit polyclonal anti-HPDE4B2 antibody (1:1000; Santa Cruz. Biotech) and secondary HRP-conjugated anti-rabbit antibody (1:1000; Santa Cruz Biotech).
Example 7: Purifying the recombinant hPDEs
The recombinant hPDE4B2 expressed in Dictyostelium discoideum was purified using Ni-NTA affinity column as per manufacturer's protocol (Amersham). Briefly, the samples were prepared as described above in Example 6 and filtered sequentially through 0.45µ and 0.22ja filters. The samples were loaded at 0.25 ml to 0.35 ml per minute and washed with 6 to 10 column volume of wash buffer (20mM Sodium Phosphate, 0.5 M NaCl and 40 mM Imidazole pH 7.4). A gradient of varied imidazole concentration from 0 to 500 mM was run and the protein was eluted at 50% concentration gradient (Fig. 4a). The eluted fraction number (23 to 27) showed partially purified hPDE4B2 by SDS-PAGE that was confirmed by western blot using anti-PDE4B2 antibody (Fig. 4b). Approx. 1 mg of recombinant hPDE4B2 was purified using this method, indicating production of sufficient amount of protein needed for in-vitro high-throughput assays.
The recombinant hPDE4B2 was also expressed in mammalian HEK293 cells as described before (Bora et al., 2007). The cell density of maximum IX 105 cells/ml was achieved in the cell stacks. The cells were collected over a period of one month from four cell stacks. Approx. 10 cells were lysed and the cell lysates was loaded on PDE4B2 antibody coupled NHS-activated sepharose column for immunoaffinity purification. The fraction number 27 to 33 were pooled (Fig. 4c) and subjected to SDS-PAGE followed by western blot analysis. The expected band size of-67 kd was observed as shown in Fig. 4d. We were able to partially purify 50jo,g of hPDE4B2 using this method. Thus, 20-fold increase in yields were observed when the protein was expressed in Dictyostelium AX3 cells compared to mammalian HEK293 cells, that too, in a much reduced time, labour and cost.
(Figure 4 Removed) Fig.4
Example 8: Testing the activity of purified recombinant hPDEs by cAMP assay using Discovcrx kits.
Determination of Enzyme kinetics: For enzyme kinetics, the PDE assays were conducted with 10-15 different cAMP concentration over a range of 0.1 to 100 µM. Kinetics was determined by Michaelis-Menten parameters in Graph Pad prism using a non-linear regression analysis. Statistical comparison was done using F test/ student t test. For inhibitor studies, ICso was determined over a range of inhibition concentration from 1 nM to 1 OnM unless otherwise stated. Enzyme assay were performed using cAMP Hit Assay kit (Discoverx). Samples were diluted to ensure that the kinetics remained in the linear range and that no more than 20% of the substrate was consumed. Briefly, hPDE4B2 cell lyaste was added at different concentrations in a 96 well dish. cAMP was added to the final concentration of 1 µM and PDE4B2 inhibitor was added at lOµM concentration. 0.05 M Tris pH 7.4, 0.5 mM MgCl2, and 1 mM EGTA buffer made final volume to 100 ul. The reaction mixture was incubated at 30°C for 1 h at 120-140 rpm shaker. 15 ul of the reaction mix was added to 10 ul lysis buffer and incubated at room temperature for 1 h in dark. 13 µl Fluorescent substrate (FL/ED) was added and incubated at room temperature for 1 h. lOul EA reagent was added and the plates was further incubated at room temperature for 1 h. The reading was taken in fluorimeter at 530 nm excitation and 610-emission wavelength. Three parameters were used to compare the activity of hPDE4B2 expressed in Dictyostelium vs mammalian system. Interaction at the catalytic sites was assessed by determination of Km for
cAMP and ECso for activation by Mg2+. Inhibitor interaction was determined by employing a panel of eight inhibitors to measure the affinity for each compound. At 1 µM cAMP, purified hPDE4B2 from Dictyostelium cells showed a 20-fold increase in cAMP hydrolyzing PDE activity, as compared with non-expressing cells. The specific activity of hPDE4B2 was determined to be 66 pmol/min/mg which is comparable to specific activity of hPDE4B2 expressed in mammalian HEK293 cells. To further characterize its enzymatic properties, more detailed kinetic studies were performed. Lineweaver-Burk plots for hPDE4B2 expressed in both the systems were linear over a wide range of cAMP concentrations. The Km of recombinant hPDE4B2 as determined by Michaelis-Menten kinetics, were 1.25 µM ± 0. 02 in Dictyostelium and 3.3 µM ± 0.5 in mammalian cells (Fig. 5).

(Figure 5 Removed)
Fig. 5 Michaelis-Menten kinetics of expressed recombinant hPDE4B2
Thus, HPDE4B2 expressed in both systems showed Km values within the previous reported range of 1-3 uM. The interaction with Mg2+was not altered for PDE4B2 in the two systems and the EC50 value was similar to previously reported values in mammalian cells. These, studies suggest that the catalytic domain of hPDE4B2 expressed in Dictyostelium maintains the right conformation for interaction with substrate and bivalent cations.
Biological Activity Data
Sensitivity of hPDE4B2 to several inhibitors
An obvious approach to characterize the physiological role of hPDE4B2 is to block its activity using specific drugs. Therefore, we used a wide range of commonly available PDE inhibitors to determine specificity of hPDE4B2 expressed in Dictyostelium cells. The spectrum of inhibitors tested in this study included nonselective compounds as well as selective inhibitors targeting members of other known PDE families. The result of the inhibitor studies are summarized in Table 1. Three PDE4-specific inhibitors roflumilast (phase III clinical), cilomilast (pre-registeration stage) and rolipram (failed in clinical) showed high sensitivity to hPDE4B2 expressed in Dictyostelium (Lipworth, 1995; Lugnier, 2006; Dastidar et al., 2007). The ICso of roflumilast was 7nM ± 0.5, rolipram was 1.0 µM ± 109 and cilomilast was 70 nM ± 10, as represented by average of three separate experiments with two independently enzyme preparations. Several other inhibitors used in this study (Table 1) did not effect PDE4B2 hydrolysis of cAMP, even when applied at concentrations that exceeds the IC50 values for other PDEs by 100-fold. The IC50 values of different inhibitors were also studied in parallel experiments with hPDE4B2 expressed in mammalian cells and compared to the ones obtained from Dictyostelium expressed hPDE4B2. The IC50 of roflumilast, rolipram and cilomilast were 8.8 nM ± 0.92, 1.5 µM ± 20.9 and 352 nm ± 35, respectively (Table 1). As expected, hPDE4B2 expressed in HEK293 did not show specific interaction with IBMX, dipyridamol, EHNA, cilostamide and Zaprinast. These studies clearly indicate that PDE4B2 expressed in Dictyostelium discoideum is pharmacologically active with similar properties to those expressed in mammalian cells.Table 1
(Table 1 Removed)