Field of the Invention
This invention relates to the process of producing biologically active human photoreceptor cGMP- phosphodiesterase PDE6α in a bacterial expression system. The method is directed to the over expression of PDE6α protein as inclusion bodies in bacteria and a method of purification to produce a biologically active PDE6α in a single step.
Background of the Invention
Purine cyclic nucleotides viz. cAMP and cGMP are ubiquitous second messenger molecules that are responsible for the phosphorylation and activity of various proteins. Purine cyclic nucleotides are generated at the cytosol surface of the plasma membrane through the action of adenylate cyclases (Biochem. J. 370 (2003) 1-18). The only way to inactivate these cyclic nucleotides is hydrolysis of the phosphodiester bond through the action of Phosphodiesterase (PDE) enzymes (Biochem. J. 370 (2003) 1-18). This hydrolysis converts the cyclic forms to corresponding 5'-mononucleotides (./. Biol. Chem. 237 (1962) 1244-1250).
There are 11 isoforms within the PDE family {Biochem. J. 370 (2003) 1-18). Each member is characterized by specific substrate selectivity and it's response to various activators and inhibitors. The catalytic domain of individual members shows modest sequence conservation. The individual members have a distinct tissue distribution that usually is very complex. Several of these members have been selectively targeted for development of therapeutic agents for various indications. The most prominent examples are PDE5 for erectile dysfunction {Urology 60 (suppl. 2B) (2002) 4-113) and PDE4 for inflammation (Curr. Pharm. Des. 8 (2002) 1255-1296). The population of PDEs in the photoreceptors is almost exclusively restricted to the PDE6 family. The last component of the cGMP-related phototransduction machinery is PDE6, the enzymes that degrades cGMP in photoreceptors. The hydrolysis of cGMP by the PDE6s is the final step of signal amplification in the retinal phototransduction cascades. The molecular machinery of the mammalian phototransduction machinery combine to create a complex cascade in which cGMP and Ca2+ strictly interact to convert light in terms of photons or external energy to internal messages of electrical impulses. Thus, high levels of cGMP during the dark state due to weak activity of PDE6, ensures a high concentration of Ca2+ and in presence of light, activated PDE6 degrades cGMP, thereby leading to a chain of depolarization/polarization events inhibitor (Biochem. Pharma. 68 (2004) 867-873). Nevertheless, the main concern in the development of PDE inhibitors as therapeutic agents is the selectivity for a particular PDE. To avoid the side/unwanted effects of a new chemical entity (NCE), for example, inhibition of PDE 6 would lead to a rise in cGMP levels even in presence
of light leading to a severe imbalance in the phototransduction machinery. It is important that an NCE should affect/inhibit only a specific PDE and not any other. Thus, for PDE selectivity evaluation in drug discovery, availability of individual PDEs is of paramount importance.
PDE6 is a phosphodiesterase that is found only in rod and cone cells of the eye. This is the exclusive isoform of PDEs that is present in the eye. The rod PDE6 holoenzyme is a heterotrimer composed of an and two  subunits. The cone PDE6 holoenzyme is a heterodimer composed of two a'and two y subunits. The α, α' and  subunits are large proteins of nearly 99kDA each where as y subunit is a small protein of 1 lkDA. The a and  subunits are catalytic units where as y subunits function as an internal enzyme inhibitor (Biochem. Pharma. 68 (2004) 867-873). Three independent genes code for these catalytic units in human and their corresponding cDNAs have been cloned and sequenced (J. Biol. Chem. 279 (2004) 19800-19807; J. Biol. Chem. 277 (2002) 25877-25883; FEBS Lett. 381 (1996) 149-152; Genomics 6 (1990) 272-283). The cDNAs for catalytic units of PDE6 have also been cloned in mouse and bovine (Genomics 6 (1990) 272-283; FEBS Lett. 278 (1991) 107-114; J. Biol. Chem. 265 (1990) 12955-12959; Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 293-297). Since PDE6 holoenzyme has two catalytic subunits it appears that both the catalytic units of PDE6 need to be expressed together for a functionally active enzyme. Several studies have been done on expression of catalytically active mouse or bovine recombinant PDE6 in yeast, bacteria, insect cells and mammalian cells (J. Biol. Chem. 267 (1992) 8458-8463; Proc. Natl. Acad. Sci. 90 (1993) 9340-9344; Mol. Vis. 9 (2003) 80-86; J. Biol. Chem. 269 (1994) 3265-3271). The catalytic activity of PDE subunits individually expressed in unicellular systems has been insignificant in most studies. Nevertheless, in mammalian cells the individually expressed PDE subunits showed some activity (Proc. Natl. Acad. Sci. 90 (1993) 9340-9344; Mol. Vis. 9 (2003) 80-86). There is also a single report on functional expression of human PDE6 in baculovirus expression system (Biochem. Pharma. 68 (2004) 867-873). They have indicated that the inhibitor profile of the native PDE6 enzymes differs from those shown by the recombinant PDE6 enzymes.
Rod and cone cGMP-phosphodiesterases (PDE6) serve as key effectors enzymes in the visual transduction cascade of vertebrate photoreceptor cells. Progress in understanding the structure and function of PDE6 has been hindered by difficulties in developing an efficient functional expression system for PDE6. Moreover, the availability of purified PDE6 will assist in screening the specificity of isozyme selective PDE inhibitors in drug discovery. Although rod PDE6 essentially contains two catalytically active subunits, a and p, but here we show that expression of even one catalytic unit (a) in bacteria yielded active enzyme. None of the prior art suggests the expression of functionally active human photoreceptor cGMP- phosphodiesterase, PDE6, in bacteria.
Summary of the Invention
A method is provided for producing biologically active PDE6 in a bacterial expression system at high levels. The method comprises:
a) sub cloning PDE6 cDNA into a bacterial expression system (recombinant Escherichia coli plasmid vector) and transformation of the same to obtain a recombinant clone operatively linked to a DNA sequence encoding an additional amino acid sequence at the N terminus of the cloned protein which facilitates purification of protein;
b) culturing the recombinant bacteria to induce protein production;
c) harvesting and lysing the cells to produce a pellet fraction containing the recombinant protein;
d) solubulizing recombinant PDE6 protein from the insoluble fraction;
e) adsorbing, refolding and purifying the recombinant PDE6 protein in a single step by chromatography methods to get biologically active PDE6.
In another embodiment the additional sequence comprises a cluster of positively charged amino acid residue for example, histidine, arginine, lysine and mixtures thereof, The added sequence comprises histidine residues, and particularly 6 to 10 histidine residues. The use of this sequence allows one- step purification using a metal affinity or chelating column such as a nickel (II) nitrilotriacetic acid agarose or a metal chelating sepharose column charged with nickel (II).
Detailed Description of the Invention
The present invention encompasses method of producing biologically active human photoreceptor cGMP- phosphodiesterase PDE6 in a bacterial expression system.
The production method of this invention for obtaining the biologically active human photoreceptor cGMP- phosphodiesterase PDE6α is comprised of introduction of human rod PDE6α gene into a plasmid vector, the vector is introduced into living E.coli cells, and a large amount of PDE6α enzyme is produced and isolated from the culture, which is then purified to get the biologically active human PDE6α.
PDE6α is expressed as protein containing additional amino acid sequence that facilitate purification of the polypeptide, particularly by cation or anion exchange, affinity, or immunoaffinity chromatography. This is achieved using a vector, which puts the desired additional amino acid sequences of desired lengths. Examples of such additional sequences include without limitation clusters of charged amino acids from 3 to 15 residues in length, this
additional sequence comprises a cluster of positively charged amino acids selected from histidine, arginine, lysine and mixtures thereof which facilitates rapid and inexpensive purification by, for example, metal chelation chromatography. The preferred added sequence comprises histidine residues, and particularly 6 to 10 histidine residues.
The expression vector used in the present invention includes chromosomal- and episomal- derived vectors, e.g., vectors derived from bacterial plasmids. Among vectors used in the present invention include but not limited to pQE30, pQE60 or pQE9 available from Qiagen; the representative of bacterial cell capable of being transformed and then expressing the protein include but are not limited to E. coli, bacillus species, lactobacillus species and streptococcus species.
The expression vector includes at least one selectable marker. Such markers include but not limited to ampicillin, kanamycin or tetracycline or mixture(s) thereof resistant gene for culturing in E. coli and other bacteria.
For culturing, recombinant bacteria from the glycerol stock was inoculated in Luria-Bertani medium (LB medium) that was supplemented with ampicillin and kanamycin and was grown overnight. Overnight culture was diluted in fresh LB medium and growth was allowed to occur until O.D.600 reached 0.8. At this stage, IPTG (isopropylthio-D-galactoside) was added. In this system, the addition of IPTG (isopropylthio-D-galactoside) to the culture induces the lac operon system, which in turn allows for the transcription and translation of the target DNA. After induction was over, the bacterial cells were pelleted down by centrifugation. The cell pellet was suspended and lysed and then the cells were incubated on ice. Inclusion bodies including the recombinant His-tagged PDE6 were separated from soluble proteins by centrifugation. To wash off the phospholipids and carbohydrate moieties from the inclusion bodies, the pellet was treated with buffer followed by incubation on ice. The pellet was separated from the wash buffer by centrifugation. Following centrifugation, the pellet was suspended by vortexing in denaturing buffer followed by sonication. The suspension was incubated on ice and then centrifuged. The supernatant was treated with DNase.
Other solidified or liquid mediums which can support growth and reproduction of E. coli is useful in cultures are also included for practicing the method of the invention, (LB, 2YT, nutrient broth, M9 media, soft agar media or chemically defined media, etc). Numerous suitable bacterial media are known to those skilled in the art. Typical media include both minimal and rich media. Minimal media include mixtures of magnesium sulfate and a carbon source, typically sugar or glycerol, with M9 medium containing disodium phosphate, monobasic potassium phosphate, ammonium chloride, sodium chloride and optionally calcium chloride, M63 medium containing ammonium sulfate, monobasic potassium phosphate, and ferrous
sulfate; or a medium containing ammonium sulfate, monobasic potassium phosphate, dipotassium phosphate, and sodium citrate. Thiamine, Casamino acids (Difco, Detroit, MI 48232-7038), L-amino acids, and antibiotics may be added, if required. Rich media include H medium containing tryptone and sodium chloride; Luria broth containing tryptone and sodium chloride; Luria-Bertani medium containing tryptone, yeast extract, sodium chloride, NZC broth containing NZ amine A (Hunko Sheffield), sodium chloride, magnesium chloride and Casamino acid (Difco); Superbroth containing tryptone, yeast extract, sodium chloride, and sodium hydroxide; tryptone broth containing tryptone and sodium chloride; TY medium containing tryptone, yeast extract, and sodium chloride; and TYGPN medium containing tryptone, yeast extract, glycerol disodium phosphate and potassium nitrate.
Purification of PDE6α can be achieved using methods known in the art. Purification from the soluble fraction can be achieved in a single step using for example, metal chelation chromatography, affinity chromatography, immunoaffinity chromatography, and anion and cation exchange chromatography. It is to be understood that the purification method used will depend upon the additional sequence that is added. For example, when 6-10 histidine residues are fused to the PDE6α sequence, affinity chromatography or metal chelation chromatography and preferably Ni(II)nitrilotriacetic acid agarose chromatography (Ni-NTA) can be used to purify the recombinant PDE6α.
The recombinant protein was purified from the bacterial cell lysate by affinity chromatography. The protein was recovered in a denatured state after lysis of the bacterial cells. To prevent aggregation of the protein upon refolding, a strategy was used by adsorbing the denatured protein molecules to a solid support, thus effectively separating the individual protein molecules from each other during refolding. Non-covalent (reversible) adsorption of denatured proteins to immobilized metal-ion affinity chromatography (IMAC) media was used for the purpose of refolding using a three-buffer system (binding buffer containing urea, binding buffer without urea and elution buffer). The column was equilibrated with binding buffer containing urea. The sample dissolved in urea was adsorbed to the MAC media and the urea concentration in the column was gradually decreased by the introduction of binding buffer without urea which acted as the refolding buffer leading to gradual refolding of the adsorbed protein molecules. After all the denaturant was washed out of the column, the refolded and still adsorbed protein molecules were released by the addition of elution buffer containing imidazole. This process allows higher yield over other conventional two buffer systems purification and furthermore, the process was minimized by coupling the binding, refolding and elution steps as a single column method.
The invention will be better understood by reference to the experimental details which follows, but those skilled in art will readily appreciate that the specific experiments detailed are only illustrative, and are not meant to limit the invention as described herein, which is defined by the claims which follows thereafter.
Materials and methods
Human rod PDE6α (GenBank Accession No. M26061, Prolab, India)
pQE30 vector (Qiagen)
IPTG (isopropylthio-,-D-galactoside) (Sigma-Aldrich, USA)
IX protease inhibitor-EDTA free (Roche Applied Sciences, USA),
Lysozyme (lmg/ml, final concentration) (Sigma Aldrich, USA)
Denaturing buffer (Roche Applied Science, USA)
Ni-NTA column (Amersham Biosciences, USA)
Cartridge filter (Millipore, USA)
Biologic Maximizer 20 System (Bio-Rad, USA)
Bio-Frac fraction collector (Bio-Rad, USA).
Example 1 Cloning of human rod PDE6α
The 2.6kb Human rod PDE6α (GenBank Accession No. M26061 Jrolab, India).) cDNA gene was cloned in the pQE30 vector (Qiagen) at Smal site by standard protocol. This vector puts a 6X His-tag at the N terminus of the cloned protein. The recombinant clones were confirmed by restriction digestion.
Example 2 Expression and purification of recombinant human rod PDE6α
The recombinant bacteria from the glycerol stock were inoculated in 50 ml LB medium that was supplemented with l00µml ampicillin and 25µg/ml kanamycin and was grown overnight at 37°C. Overnight culture was diluted 1:20 in 1 liter fresh LB medium and growth was allowed to occur until O.D.600 reached 0.8. At this stage, ImM of IPTG (isopropylthio-,-D-galactoside) was added to induce protein production. The induction was done for two hours at 37°C. Following induction, the bacterial cells were pelleted down by centrifuging at 4000 rpm for 30 min at 4 C. The cell pellet was suspended and lysed in 20ml lysis buffer (1 mM Tris base
pH-7.4, 3 mM NaCl, 10 mM DTT, IX protease inhibitor-EDTA free, 1% Triton X100, lysozyme (lmg/ml, final concentration).
To ensure an efficient lysis, the cells were subjected to 6 cycles of quick freeze thaw (liquid nitrogen / water bath 37 °C) and then the cells were incubated on ice for 45 min. Inclusion bodies including recombinant His-tagged PDE6 were separated from soluble proteins by centrifugation at 30,000 rpm for 30 min at 4 C. To wash off the phospholipids and carbohydrate moieties from the inclusion bodies, the pellet was treated with 40 ml wash buffer (IX PBS without Ca and Mg and supplemented with 25% sucrose and 1% Triton X-100) followed by 10 min. incubation on ice. The pellet was separated from the wash buffer by spinning at 15,000rpm for 15min at 4 C. Following centrifugation, the pellet was suspended by vortexing in 20 ml of denaturing buffer (50mM Tris-Cl, pH 7.4, 1% Tween-20, l0mM P-Mercaptoethanol, 8M Urea, 0.5 M NaCl, 10% glycerol), followed by 8 cycles of sonication using 20 sec burst at 150-170 W and 1 min cooling period between each burst. The suspension was incubated on ice for 1 hour and then centrifuged at 30,000rpm for 30min at 4 C. The supernatant was treated with DNAse 1, 2 units per ml of denaturing buffer for 8-10 hrs at 4 C. Before loading it on to the Ni-NTA column, the supernatant was centrifuged at 10,000 rpm for 15 mins and filtered with 0.2 µ, cartridge filter to remove any particulate matter.
The Ni-NTA column was equilibrated with the binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, 8M Urea, pH 7.4) using the Biologic Maximizer 20 System. The supernatant was then loaded on to the column using the flow rate of 0.35 ml/min and the column was washed with binding buffer at 1 ml/min till the effluent O.D came to a minimum (O.D280 < 0.001). The bound denatured His-tagged protein was allowed to refold by running a reverse urea gradient (100% to 0%) in binding buffer over 6 ml at a flow rate of 1 ml/min. Further washing with binding buffer (without any urea) was done to eliminate any non-specific binding. The refolded His-tagged protein was eluted with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 250 mM imidazole, pH 7.4). The fractions were collected using Bio-Frac fraction collector.
Immunoblotting analysis
Following purification, the protein samples were run in 10% SDS-PAGE mini-gels and proteins were transferred to nitrocellulose membrane by semi-dry method. The proteins were detected using a mouse monoclonal anti-polyhistidine antibody and developed by using DAB with metal enhancer. Purified fractions showed molecular mass of about 99 KDa (Fig. 3).
PDE6 activity assay
The PDE6 activity was assessed by HitHunter™ cGMP assay kit. The assay buffer had 50 mM Tris HCl (pH 7.5), 8.3 mM MgCl2 and 1.7 mM EGTA. Briefly, 0.2 µM cGMP was mixed with varying amounts (10-50 ul) of purified PDE6 in total of 90 µ1 reaction. For negative control, elution buffer was used instead of PDE6. Reaction was incubated at 37°C for 20 minutes. Then 30 ul aliquot was tested for non-hydrolyzed cGMP levels as per the kit protocol with slight modification. Each aliquot was tested in duplicate in a 96 well flat bottom plate. The anti-cGMP antibody was diluted 1:2 in PBS before use. The samples were mixed with 20 µl of diluted anti cGMP antibody and 20 µl of cGMP-ED reagent before incubating for 60 minutes at room temperature (24°C). Following this incubation, 20 ul of cGMP-EA reagent was added and incubation was continued for additional 60 minutes. Subsequently, 30 µl of the luminescent substrate was added and counts were taken in a luminometer after two hours of incubation at room temperature.
The elution buffer did not result in any hydrolysis but by using 10, 20, 30, 40, 50 µl aliquots of PDE6 resulted in a dose-dependent hydrolysis of cGMP (Fig. 4).
Results Expression and purification of rod PDE6α
Expression studies indicated that 2 hours of induction with IPTG was inducing good amounts of the enzyme PDE6oc. However, most of the protein was in the pellet i.e. insoluble form (Fig 1). To extract the protein from the inclusion bodies, an elaborate purification protocol was followed. The protein eluted from the Ni-NTA column as a single peak (Fig. 2). Purified fractions were checked by immunoblotting and a protein with the expected molecular mass of about 99 KDa was detected (Fig. 3).
Enzyme activity of the recombinant protein
Purified PDE6 was assayed for its ability to hydrolyze cGMP. The elution buffer did not result in any hydrolysis but by using 10, 20, 30, 40, 50 µl aliquots of PDE6 resulted in a dose-dependent hydrolysis of cGMP (Fig. 4).

WE CLAIM:
1. A method is provided for producing biologically active PDE6 in a bacterial expression system at high levels. The method comprises:
a) sub cloning PDE6 cDNA into a bacterial expression system (recombinant Escherichia coli plasmid vector) and transformation of the same to obtain a recombinant clone operatively linked to a DNA sequence encoding an additional amino acid sequence at the N terminus of the cloned protein which facilitates purification of protein;
b) culturing the recombinant bacteria to induce protein production;
c) harvesting and lysing the cells to produce a pellet fraction containing the recombinant protein;
d) solubulizing recombinant PDE6 protein from the insoluble fraction;
e) adsorbing, refolding and purifying the recombinant PDE6 protein in a single step by chromatography methods to get biologically active PDE6.

2. A process according to claim 1 wherein bacterial expression system, Escherichia coli plasmid vector is pQE30.
3. A process according to claim 1 wherein the additional sequence at the N terminus of the cloned protein comprises of 6 histidine residues.
4. A process according to claim 1 wherein culturing the recombinant bacteria to induce protein production was done in LB medium supplemented with ampicillin and kanamycin.
5. A process according to claim 1 wherein the one- step purification by chromatography methods was done using nickel (II) nitrilotriacetic acid agarose .