WO2006067613A1

WO2006067613A1 - Compositions and methods for expression and purification of fatty acid amide hydrolase

Info

Publication number: WO2006067613A1
Application number: PCT/IB2005/003930
Authority: WO
Inventors: Norman Yves Garceau; Leyu Wang; Kyunghye Ahn Wisner
Original assignee: Warner Lambert Co LLC
Current assignee: Warner Lambert Co LLC
Priority date: 2004-12-22
Filing date: 2005-12-14
Publication date: 2006-06-29
Anticipated expiration: 2007-06-22
Also published as: CA2592138A1; JP2008529481A; EP1831364A1

Abstract

The invention relates to nucleic acid compositions encoding truncated FAAH polypeptides, methods of expressing recombinant FAAH, and methods of purifying recombinant FAAH. The nucleic acid compositions comprise sequences that are optimized for transcription in a prokaryotic host.

Description

COMPOSITIONS AND METHODS FOR EXPRESSION AND PURIFICATION OF FATTY

ACID AMIDE HYDROLASE

FIELD OF THE INVENTION

The invention relates to compositions and methods for enhanced expression and purification of fatty acid amide hydrolase (FAAH).

BACKGROUND OF THE INVENTION

Fatty acid amides represent a growing family of bioactive lipids with diverse cellular and physiological effects (Cravatt, B. F. et al. (1995), Science 268:1506-1509); (Devane, W. A. et al. (1992), Science 258:1946-1949); (Facci, L et al. (1995), Proc. Natl. Acad. Sci. 92:3376-3380). Fatty acid amides are hydrolyzed to their corresponding fatty acids by enzymes known as fatty acid amide hydrolases (FAAH) (Ueda et al. (2000), Chem Phys Lipids 108(1 -2):107-21). FAAH is a mammalian integral membrane serine hydrolase responsible for the hydrolysis of a number of primary and secondary fatty acid amides, including the neuromodulatory compounds anandamide and oleamide. Anandamide (arachidonoyl ethanolamide) has been shown to possess cannabinoid-like analgesic properties and is released by stimulated neurons. The effects and endogenous levels of anandamide increase with pain stimulation, implying its role in suppressing pain neurotransmission and behavioral analgesia. Supporting this, FAAH inhibitors that elevate brain anandamide levels have demonstrated efficacy in animal models of anxiety. A second fatty acid amide, oleamide (9-Z-octadecenamide) has been shown to induce sleep. FAAH is highly expressed in neurons within the central nervous system where the enzyme appears poised to inactivate fatty acid amides at their presumed sites of action. The fatty acid amides are hydrophobic in nature and interact between the enzyme and cell membrane for efficient rates of substrate hydrolysis and product release. FAAH is an integral membrane protein that degrades bioactive fatty acid amides thereby terminating the signaling functions of the fatty acid amides. Defining the nature of FAAH's membrane interactions can provide a greater understanding of how FAAH participates in the regulation of fatty acid amide signaling. Given the role of FAAHs in fatty acid amide metabolism, there is a need for further characterization of this enzyme. However, obtaining sufficient quantities of purified, active enzyme for functional- and high-resolution crystallographic studies has been difficult.

Primary sequence analysis of the mouse, rat, and human FAAH proteins predicts that these enzymes are Type 1 integral membrane proteins with a single transmembrane domain and a large cytoplasmic tail containing the majority of the protein sequence (Patricelli, et al., Biochemistry, 1998 vol.37, 15177). Rat FAAH was cloned {Nature 1996, 384, 83-7) and expressed in E. coli (Biochemistry 1998, 37, 15177-15187). Human FAAH has also been cloned (US2002/0187542 A1). Human FAAH shares 82% amino acid identity with rat FAAH and the human FAAH expression in E. coli has been minimal at best, due to difficulties in obtaining soluble active human FAAH.

SUMMARY OF THE INVENTION

The present invention encompasses nucleic acid compositions encoding truncated human FAAH polypeptides, methods of expressing recombinant human FAAH in E. coli, and purifying the expressed polypeptide. In particular embodiments of the present invention set forth below, the nucleic acid sequences are optimized for transcription in a prokaryotic host.

In one aspect, the invention is an isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: a. A nucleotide sequence set forth in SEQ ID NO:1 ; b. A nucleotide sequence that is 95 to 99% identical to the nucleotide sequence set forth in SEQ ID NO:1 ; c. A nucleotide sequence set forth in SEQ ID NO:2; d. A nucleotide sequence that is at least 70% identical to the nucleotide sequence set forth in SEQ ID NO:2; e. A nucleotide sequence encoding amino acids 30-579 of SEQ ID NO: 4; and f. A nucleotide sequence encoding amino acids 32-579 of SEQ ID NO: 4.

In another aspect, the invention is a vector comprising the isolated nucleic acid described above. In another aspect, the invention is a host cell transformed with the vector described above.

In one embodiment, the invention is a bacterial host-cell transformed with the vector described above.

In another aspect, the invention is a method for expression of a FAAH protein comprising transforming E. coli host-cells with a vector comprising a nucleic acid molecule encoding a FAAH polypeptide and inducing expression of said polypeptide wherein said expression yields at least 0.13 to 50 mg of FAAH polypeptide per 1 liter of cell culture. In one embodiment, said expression yields 0.13 to 26 mg of FAAH polypeptide per 1 liter of cell culture. In another aspect, the invention is a method for the expression of a FAAH protein comprising culturing the host cells described above. In another aspect, the invention is a method of producing purified FAAH polypeptide, comprising: a. expressing a heterologous FAAH polypeptide in a host-cell, and b. isolating the membrane fraction of said host-cells by at least one ultracentrifugation step in absence of detergent.

In another aspect, the invention is a method for producing purified FAAH polypeptide, comprising: a. expressing a heterologous FAAH polypeptide in a host-cell; b. lysing said host-cells in the absence of a detergent; c. centrifuging the lysate of Step (b) at a low speed and retaining supernatant; d. ultracentrifuging said supernatant and retaining pellet; e. re-suspending said pellet in a buffer lacking detergent, f. ultracentrifuging said re-suspension and retaining pellet, g. solubilizing soluble polypeptides comprised in said pellet of Step (f) by re- suspending said pellet in a buffer comprising non-ionic detergent; h. ultracentrifuging said re-suspension of Step (g) and retaining supernatant, and i. isolating FAAH polypeptide from the supernatant of Step (h).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1. depicts human FAAH, SUMO-human FAAH and rat FAAH with greater than 98% purity as estimated by Coomassie Blue staining.

DETAILED DESCRIPTION OF THE INVENTION The present invention encompasses nucleic acid molecules comprising sequences that encode truncated human FAAH polypeptides, methods of expressing recombinant human FAAH in a prokaryotic host, and methods of purifying the expressed polypeptide. In particular embodiments of the present invention set forth below, the nucleic acid sequences are optimized for transcription in Escherichia coli (E. coli). The provided nucleic acid compositions comprise those having a deletion of the region corresponding to the nucleotides encoding the transmembrane domain of the FAAH polypeptide. That is, the expression of the nucleic acid compositions of the invention produces a functional FAAH polypeptide that lacks a membrane-spanning domain. In particular embodiments of the present invention, the nucleotide compositions are further optimized for expression in a prokaryotic host. In a particular embodiment, the prokaryotic host is £. coli. In this optimization, the eukaryotic transcriptional codons are replaced with prokaryotic transcriptional codons. In other embodiments, the nucleic acid molecules of the invention comprise a first nucleotide sequence encoding the truncated FAAH polypeptide as described herein, and a second nucleotide sequence encoding a polypeptide that enhances the expression of the FAAH polypeptide; wherein the first and the second nucleotide sequences are operably linked. Thus, the present invention encompasses a fusion protein that comprises a first truncated FAAH polypeptide and an optional second polypeptide and nucleic acids encoding these fusion proteins. It is envisioned that the second polypeptide enhances the proper folding of the truncated FAAH. In one embodiment, the second polypeptide is SUMO (small ubiquitin-related modifier). For example, see Malakov et al. (2004) 5(1-2): 75-86. In another embodiment, the nucleic acid molecules of the invention further comprise a third nucleotide sequence that encodes a polypeptide that facilitates purification of the expressed protein comprising said tag. In one embodiment, the tag is a histidine (His) tag. In such embodiments that are in the context of nucleotide sequence encoding polypeptides, "operably linked" means that the expression of the nucleic acid molecule comprising the first, second and/or third nucleotide sequences in a host-cell capable of expressing said nucleotide sequences produces a fusion protein that comprises the polypeptides encoded by the first, second and/or third nucleotide sequence. It is recognized that proteolytic cleavage sites can readily be engineered in order to optionally cleave the polypeptide encoded by the second and/or third nucleotide sequence from the FAAH polypeptide encoded by the first nucleotide sequence. It is also recognized that the first and/or second nucleotide sequence can be operably linked to the 5' or the 3' end. It is further recognized that each linkage can be optionally contiguous with the third nucleotide sequence.

Methods of enhanced FAAH polypeptide expression and purification are also provided as further described below. The methods are applicable to mammalian FAAH including those set forth herein.

The compositions and methods of the invention are useful for producing functional recombinant FAAH polypeptides. The compositions and methods of the invention are useful for producing the polypeptides in quantities that can be needed for high resolution crystallography; for screening for molecules that modulate FAAH activity, including high through-put screening of peptide- and small molecule libraries; and for facilitating structure-based FAAH inhibitor design for the treatment of disorders including anxiety and insomnia.

The nucleotide set forth in SEQ ID NO.1 encodes a truncated form of the human FAAH. That is, the transmembrane domain, amino acids 1-29 of wildtype human FAAH, are deleted and amino acids 30-579 corresponding to the cytosolic domain of human FAAH, are retained. The nucleotide sequence set forth in SEQ ID NO.2 encodes a truncated form of the FAAH polypeptide wherein the transmembrane domain, amino acids

1-29, are similarly deleted and amino acids 30-579 are retained. Furthermore, the nucleotide sequence in SEQ ID NO 2 is optimized for expression in a prokaryotic host-cell with regard to codon usage. The nucleotide sequence set forth in SEQ ID NO 2 is at least 70% identical to the sequence set forth in SEQ ID NO.1. The nucleic acid molecules of the present invention encode a functional and soluble FAAH polypeptide, in that the encoded polypeptides lack a transmembrane spanning domain and catalyze a hydrolytic conversion between soporific fatty acid primary amides and their corresponding fatty acids. This catalysis is a function of wildtype FAAH and can be tested by methods known in the art (Kathuria, S. et.al. (2003), Nature Medicine 9:76-81 ); (Patricelli, M. et.al. (1998), Biochemistry 37:15177-15187); or otherwise described in the EXAMPLES described herein.

In one embodiment, the invention is a vector comprising a nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of the nucleotide sequence of the present invention described in a) to f) as described above.

In another embodiment, the vector comprises a nucleic acid molecule encoding a histidine tag. It is recognized that the sequence encoding the histidine tag can optionally and operably be linked to the 5' or to the 3' end of the sequences encoding FAAH set forth herein. It is further recognized that a proteolytic cleavage site can be optionally and operably contained between the nucleotides encoding the histidine tag and those encoding FAAH.

In another embodiment, the invention is a vector comprising a nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of the nucleotide sequence of the present invention described in a) to f) above, and further comprising a nucleic acid molecule encoding a SUMO polypeptide sequence. The SUMO-encoding nucleotide can be optionally ligated to the 5' end of the sequence encoding FAAH. For purposes of the present invention, "SUMO" means a small ubiquitin- related modifier polypeptide. Attachment of a SUMO polypeptide to the N-terminus of under-expressed proteins enhances protein expression in E.coli. Nucleotides encoding SUMO and methods for operably linking them to desired coding sequence are available; for example, from Invitrogen (Carlsbad, CA).

In another embodiment of this aspect of the invention, the vector further comprises a promoter sequence operably linked to said nucleic acid molecule. In this and other embodiments of this aspect of the invention, in the context of operable linkage of a promoter and a polypeptide-encoding nucleotide sequences, "operably linked" means that the promoter is linked to the polypeptide-encoding nucleotide sequence such that a suitable polymerase that binds the promoter transcribes the polypeptide-encoding nucleotide sequence. Methods for such operable linkage are readily recognized by the ordinarily skilled artisan. Typically, a coding sequence ligated in the correct orientation in a multiple cloning site of a commercially available vector, is operably linked to the promoter comprised by the vector.

In another embodiment of this aspect of the invention, said promoter is a T7 promoter or a Trc promoter. In yet another embodiment, said promoter is T7 promoter. Expression plasmids containing a T7 promoter include, but are not limited to pET-9a, pET-17b, pET-11a, pET-24a-d(+), pET-28a-c(+) are available from Invitrogen (Carlsbad,

CA 92008) can be used in the practice of this invention. Plasmids pET-28a-c(+) comprise, in sequence, a T7 promoter, a ribosome binding site, restriction sites to allow insertion of the structural gene and a T7 terminator sequence. In a particular embodiment, the pET-28a(+) vector is utilized to transfect the host bacterial cell. Expression plasmids containing a Trc promoter, including but not limited to pTrcHis, can also be used in the practice of this aspect of the invention.

In one aspect, the invention is a bacterial host-cell transformed with the vector comprising the nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of a) to f) as described above.

In one embodiment, the bacterial host-cell is an E .coli strain. The E. constrains including, but not limited to BL21 , BL21-AI, BL21 (DE3), and the like, can be used in the practice of this invention. In another embodiment, the E.coli strain is BL21-AI.

In one aspect, the invention is a method for expression of a FAAH polypeptide comprising transforming E. coli host-cells with a vector comprising a nucleic acid molecule encoding a FAAH polypeptide and inducing expression of said polypeptide wherein said induced expression yields at least 0.13 to 26mg of FAAH polypeptide per 1 liter of cell culture. Methods for determining the yield is readily recognizable by the ordinary skilled artisan, and/or otherwise described in the EXAMPLES set forth herein.

In one embodiment of this invention, the expression method of the invention yields 0.13, 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22,

23, 24, 25 or 26 mg of functional FAAH protein per 1 liter of cell culture. In another embodiment, the method yields 0.13 to 26 mg of functional and soluble FAAH protein per 1 Liter of cell culture.

In one aspect, the invention is a method for the expression of a FAAH protein comprising transforming E. coli host-cells and inducing expression of a FAAH protein with a vector comprising an isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of a) to f) as described above. In one embodiment of the present invention, the induction is in the presence of 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or

300 μM IPTG. In one embodiment, the induction is further in the presence of L- arabinose. For the purposes of the present invention, "IPTG" means isopropyl thiogalactoside. In another embodiment, the induction is at a temperature of 25, 26, 27, 28, 29, or 3O⁰C. In another embodiment, the induction is for 16, 17, 18, 19, 20, 21 , 22, 23, or 24 hours. In a particular embodiment, the induction is in the presence of 100 μM

IPTG and 0.2% L-Arabinose at 25°C for 16 hours.

The transformed E. coli bacterial cells can be grown in culture media including but not limited to LB or SuperBroth. The SuperBroth media contains tryptone, sodium chloride, yeast extract, and a buffering agent. In a particular embodiment, the vectors of the invention further comprise kanamycin resistance sequences. Accordingly, in one embodiment, the transformed E. coli of the present invention is grown in SuperBroth culture media in the presence of kanamycin. In another embodiment, the concentration of the kanamycin is 30 μg/ml. It is recognized that the choice of antibiotic resistance sequences comprised by the vector and the corresponding antibiotics can be varied by the ordinarily skilled artisan.

In one aspect, the invention is a method for producing purified FAAH polypeptide, comprising: a. Expressing a heterologous FAAH polypeptide in a host-cell, and b. Isolating the membrane fraction of said host-cells by at least one ultracentrifugation step in absence of detergent.

"Heterologous FAAH polypeptide" means that the polypeptide is not native to the bacterial host-cell transformed with the nucleic acid encoding said polypeptide. In one embodiment of this aspect of the invention, FAAH polypeptide is encoded by a nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of a) to f) as described above. In another embodiment, the host-cell is a bacterial host-cell.

In one aspect, the invention is a method for producing purified FAAH polypeptide, comprising: a. expressing a heterologous FAAH polypeptide in a host-cell; b. lysing said host-cells in the absence of a detergent; c. centrifuging the lysate of Step (b) at a low speed and retaining supernatant; d. ultracentrifuging said supernatant and retaining pellet; e. re-suspending said pellet in a buffer lacking detergent, f. ultracentrifuging said re-suspension and retaining pellet, g. solubilizing soluble polypeptides comprised in said pellet of Step (f) by re- suspending said pellet in a buffer comprising non-ionic detergent; h. ultracentrifuging said re-suspension of Step (g) and retaining supernatant, and i. isolating FAAH polypeptide from the supernatant of Step (h).

In one embodiment, the isolation is done by adding said supernatant onto a cationic metal column to obtain the purified FAAH protein.

In one embodiment of this invention, the host-cell is a bacterial host-cell. In a particular embodiment of this invention, the bacterial host-cell is E.coli.

The bacterial host-cells can be lysed with a microfluidizer to disrupt the cellular membrane. In this aspect of the invention, "low speed" means a centrifugation speed that typically produces a pellet comprising cellular debris and organelles, but not the cellular membrane fraction. The particular speed selected for such low speed centrifugation may vary and is readily selectable by the ordinarily skilled artisan. In one embodiment of this invention, the low speed centrifugation is at 5000 x g.

In this aspect of the invention, "ultracentrifuging" means centrifugation of the supernatant of the low speed centrifugation step at a speed that typically produces a pellet comprising the cellular membrane fraction. The "cellular membrane fraction" as used herein, is intended as the fraction comprising membranes and integral- and associated membrane proteins thereof. By "integral membrane proteins" as used herein, is intended to mean proteins comprising at least one transmembrane domain. By "associated membrane proteins", as used herein, is intended to mean proteins that lack a transmembrane domain but are otherwise comprised by the above cellular membrane fraction. It is recognized that the FAAH polypeptides encoded by the nucleic acids of the invention are associated membrane proteins. That is, the polypeptides lack a transmembrane domain; and nevertheless are contained in the cellular membrane fraction of the ultracentrifugation step. In one embodiment of this invention, ultracentrifuging is at speeds of 50,000 to 100,000 x g. In another embodiment, the supernatant is centrifuged at 100,000 x g for 1 hour.

In another embodiment of this invention, the pellet obtained following ultracentifugation is retained and re-suspended in buffer in absence of detergent, at least one time. In a particular embodiment of this invention, the ultracentrifugation and re- suspension is repeated one, two, or three times. In another embodiment, the ultracentrifugation is repeated three times. In another embodiment, the buffer is 20 mM NaPi, pH 7.8/10O mM NaCI.

Detergent encompasses ionic, cationic, and non-ionic detergents. Non-ionic detergents include, but are not limited to Brij, Igepal, Saponin, Tergitol, Triton CF, Triton

X-100, Tween, and Tyloxapal. Detergent is added to the re-suspended pellet to release the membrane bound protein fractions. In one embodiment, the non-ionic detergent is Triton X-100.

In one embodiment of this invention, isolation of said FAAH polypeptide following ultracentrifugation comprises column chromatography. In a further embodiment, the FAAH polypeptide is fused to a histidine tag, and the column chromatography comprises utilizing a column comprising beads charged with a cation. The metal cation has an affinity towards the Histidine His-tag. Thus, the His-tagged human FAAH polypeptide is separated from the nascent bacterial membrane proteins and is retained on the metal cation column. Following this isolation, the tagged protein is readily eluted from the column.

In another embodiment of this aspect of the invention, the cation is nickel. Cationic metals include but are not limited to nickel or cobalt.

The present invention is described in further detail in the following non-limiting EXAMPLES. EXAMPLES

EXAMPLE 1. Cloning and Construction of hFAAH plasmids

Known molecular biological techniques are utilized to carry out the cloning and construction of constructs described herein. Such techniques are referred to, for example, in Davis et al., Basic Methods in Molecular Biology, Elsevir Sciences Publishing, Inc., New York, NY.1986; Hames et al., Nucleic Acid Hybridization, IL Press, 1985; Molecular Cloning, Sambrook et al., Current Protocols in Molecular Biology, Eds. Ausubel et al., John Wiley and Sons; Current Protocols in Human Genetics, Eds. Dracopoli et al., John Wiley and Sons; Current Protocols in Protein Science, Eds. John E. Coligan et al.,

John Wiley and Sons; and Current Protocols in Immunology, Eds. John E. Coligan et al., John Wiley and Sons).

PTrcHis A-hFAAH (encoding human (h) FAAH amino acids 30-579) The following cDNAs were custom-synthesized and subcloned into a pUC119 vector

(Blue Heron Biotechnology (Bothell, WA)). The E. coli codon optimization was carried out by using an optimization algorithm (Blue Heron Biotechnology (Bothell, WA)).

- pUC119-hFAAH (encoding hFAAH, amino acids 1-579): A cDNA containing the E. coli codon optimized DNA sequence of hFAAH (encoding amino acids 1-579) with a

5'-Xho I site, 3' stop codon , and 3'-EcoR I site. (SEQ ID NO: 6).

- pUC119-hFAAH (encoding hFAAH, amino acids 30-105): A cDNA containing the E. coli codon optimized DNA sequence of hFAAH (amino acids 30-105) with a 5'-Xho I site and 3'- Hind III site (SEQ ID NO: 7).

The pUC119-hFAAH (encoding hFAAH, amino acids 1 -579) was digested with Xho I - EcoR I and the insert was subcloned into a Xho I - EcoR l-digested pTrcHis A vector (Invitrogen, Cat # V360-20) to generate pTrcHis A-hFAAH (encoding amino acid 1 -579). The pTrcHis A-hFAAH vector was digested with Xho I-Hind III, and the resulting Xho I-

Hind III (approximately 4.5 kb) and Hind Ill-Hind III (approximately 1.5 kb) pieces were ligated with the Xho I-Hind III fragment (225 bp) generated from digesting the pUC119- hFAAH (encoding amino acids 30-105) construct with Xho I-Hind III to generate an NH₂- terminally His-tagged pTrcHis A-hFAAH (encoding amino acids 30-579).

pET28a-hFAAH (encoding hFAAH, amino acids 32-579)

The human FAAH construct for subcloning into the prokaryotic expression vector pET28a(+) (Novagen, Catalog # 69864-3) was generated by PCR from the pTrcHis A- hFAAH (amino acids 30-579) construct using the following primers: sense primer, 5'- GGAATTCCATATGTCAGGTCGTCGTACCGCACGTG-3' (SEQID NO:8) ; and antisense primer, δ'-CCGCTCGAGTTATGAGGATTGTTT TTCCGGAGTCAT-S' (SEQ ID NO: 9). The resulting PCR product was digested with Nde I-Xho I, and subcloned into a Nde I- Xho I -digested pET28a(+) vector to generate an NH₂-terminally His-tagged pET28a- hFAAH (encoding amino acids 32-579).

pET-SUMO-hFAAH (encoding hFAAH, amino acids 30-579)

The human FAAH construct for subcloning into the prokaryotic expression vector pET- SUMO (Invitrogen, Catalog #K300-01) was generated by PCR from the pET28a-hFAAH

(amino acids 32-579) construct using the following primers: sense primer, 5'- CGTTGGTCAGGTCGTCGTACCGCACG-3' (SEQID NO:10); and antisense primer, 5'- TTATGAGGATTGTmTCCGGAGTC-3' (SEQID NO:11). The resulting PCR product was subcloned into a pET-SUMO vector by TA cloning according to the manufacturer's instructions to generate an NH₂-terminally His- and SUMO-tagged pET-SUMO-hFAAH

(encoding human amino acids 30-579).

Summary of Human FAAH Constructs: pTrcHis A-hFAAH encodes hFAAH, amino acids (30-579): MGGSHHHHHHGMASMTGGQQMGRTLYDDDDKDRWGSELE — hFAAH

pET28a-hFAAH encodes hFAAH, amino acids (32-579): MGSSHHHHHHSSGLVPRGSHM— -hFAAH

pET-SUMO-hFAAH encodes hFAAH, amino acids (30-579):

MGSSHHHHHHGSGLVPRRGSASMSDSEVNQEAKPEVKPEVKPETHI NLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDME DNDIIEAHREQIGG — hFAAH

"----" Does not represent amino acid positions. Each indicated hFAAH position is contiguous with the specifically indicated leading amino acid sequence comprising

HHHHHH.

EXAMPLE 2. Expression of Truncated hFAAH "RT" describes room temperature which is typically 25°C ± 3°.

The pET28a-hFAAH construct encoding amino acids corresponding to amino acids 32- 579 of wild-type human FAAH was expressed in E. coli BL21-AI. The cultures were grown in SuperBroth media in the presence of 30 μg/ml kanamycin at 37 ⁰C and induced at OD₆₀₀ of 0.6 - 0.65 with 100 μM IPTG and 0.2% L-arabinose at RT or 30 ⁰C. Under the conditions of this EXAMPLE, an optimal expression of human FAAH was obtained after 20 hours of induction at RT.

EXAMPLE 3. Expression of the Truncated hFAAH utilizing SUMO Construct The pET-SUMO-hFAAH construct encoding amino acids corresponding to amino acids

30-579 of wildtype human FAAH was expressed in E. coli BL21 -Al. The cultures were grown in SuperBroth media in the presence of 30 μg/ml kanamycin at 37 ⁰C and induced at OD₆₀₀ of 0.6 - 0.65 with 100 μM IPTG and 0.2% L-arabinose at RT. Under conditions of this EXAMPLE, an optimal expression was obtained at 16 hours.

For producing time courses, 10 ml cultures were taken out at desired time points and pelleted at 5000 x g. The cells were washed by re-suspending in 10 ml PBS and collected by centrifugation at 5000 x g. The cells were re-suspended in 0.5 ml buffer A (20 mM NaPi, pH 7.8/100 mM NaCI) containing 1% Triton X-100, and lysozyme was added to a final concentration of 1 mg/ml. After incubation on ice for 30 minutes, the lysate was sonicated with 10 second pulses and centrifuged at 10,000 x g for 30 minutes. The resulting supernatant was used for analyzing the FAAH expression by the FAAH activity assay set forth below or by Western blot.

FAAH Activity Assay

The FAAH assay was carried out as previously described (Omeir, R. et al. (1995) Life

Sciences 56: 1999-2005) with several modifications. The typical reactions were carried out in polypropylene tubes in a total volume of 200 μl. To a reaction mixture (165 μl) containing NaPi, pH 7.4 and EDTA, was added 15 μl of [3H]anandamide (40,000 cpm) dissolved in 67% ethanol. For the initiation of the enzyme reaction, a solution of 20 μl of FAAH diluted in a buffer containing 20 mM NaPi, pH 7.4 /1 mM EDTA/0.5% Triton X-100 was added. The final concentrations of NaPi, pH 7.4, EDTA, Triton X-100, and substrate were 50 mM, 1 mM, 0.075%, and 10 μM, respectively. After incubation at 37 ⁰C for 1 hour, the reaction was stopped and the products were separated by organic solvent extraction (400 μl, choloroform: methanol, 1 :1). The mixture was vortexed thoroughly and centrifuged for 10 min at 10,000 x g. The aqueous layer (200 μl) containing

[3H]ethanolamine was quantitated by the scintillation counter.

EXAMPLE 4. Purification of hFAAH

Human FAAH plasmid, pET28a-hFAAH, was transformed into the E. co// BL21-AI strain. Two-liter cultures of the freshly transformed expressing strains were grown in

SuperBroth media in the presence of 30 μg/ml kanamycin at 37 ⁰C. At OD₆₀₀ of approximately 0.12, the cultures were transferred to RT and induced at OD₆₀₀ of 0.6 - 0.65 with 100 μM IPTG and 0.2% L-arabinose for 20 hours at RT. All operations below were at 4 °C unless otherwise noted. The cells were then harvested by centrifugation at 5000 x g and the cell pellets were washed by re-suspending in 700 ml of PBS and collected by centrifugation at 5000 x g. At this point, the cell paste is optionally frozen and stored at - 80 ⁰C until needed. The cells were re-suspended in 100 ml of buffer A (20 mM NaPi, pH 7.8/100 mM NaCI) with stirring. After adding 500 units of benzonase, the cell suspension was stirred for an additional 15 minutes before lysing the cells by passing through the microfluidizer (Microfluidics Corp., Newton, MA) according to manufacturer's instructions. The cell debris was removed by centrifugation at 5000 x g for 20 minutes and the supernatant was centrifuged at 100,000 x g for 1 hour. The resulting pellet was re-suspended in 100 ml buffer A and centrifuged again at 100,000 x g for 1 hour. After repeating the last step two more times, the pellet was re-suspended in 80 ml of buffer B (20 mM NaPi, pH 7.8/500 mM NaCI/1% Triton X) and centrifuged at 100,000 x g for 1 hour and the resulting supernatant was loaded at 0.5 - 1 ml/min onto a 1 ml Ni column that has been equilibrated with buffer B. The column was washed with 20 ml of buffer B at 0.2 ml/min. The column was further washed with 10 ml of buffer B containing 20 mM imidazole and then with 10 ml of buffer B containing 50 mM imidazole at 0.2 ml/min. Elution was achieved with a 20 ml gradient (buffer B, 50 - 200 mM imidazole) at 0.2 ml/min. Fractions of 1 ml were collected. Peak fractions were dialyzed immediately against buffer C (20 mM NaPi, pH 7.8/500 mM NaCI) overnight. SDS-PAGE (4-20% gradient) visualized by Coomassie Blue staining revealed a predominant single band corresponding to the expressed hFAAH as shown in FIGURE 1 (labeled as hFAAH).

EXAMPLE 5 - Purification of SUMO-hFAAH

For pET-SUMO-hFAAH, two-liter cultures were prepared using the pET-SUMO hFAAH in the same way as described above for that of pET28a-hFAAH except that the induction was carried out for 16 hours instead of 20 hours. SUMO-FAAH was purified using the same procedure as described above for that of pET28a-hFAAH with the following modifications. The elution from a 5 mi Ni column was carried out using a 40 ml gradient (buffer B, 50 - 350 mM imidazole). SDS-PAGE (4-20% gradient) visualized by Coomassie Blue staining revealed a predominant single band corresponding to the expressed SUMO-fused hFAAH as shown in FIGURE 1 (labeled as SUMO-hFAAH).

Under the conditions described in the above methods, nucleotide encoding human FAAH polypeptide operably linked to the pTrcHis A vector promoter yielded 0.13- 0.23 mg FAAH per liter of culture. A nucleotide encoding human FAAH polypeptide operably linked to the pET28a vector promoter yielded 2.5-4.5 mg FAAH per liter of culture. A nucleotide encoding human FAAH polypeptide operably linked to a SUMO fusion protein and a pET vector promoter yielded 14-26 mg FAAH per liter of culture.

EXAMPLE 6 - Cloning, Expression and Purification of a truncated rat (r) FAAH: pTrcHis A-rFAAH (amino acids 32-579): The rat FAAH cDNA that encodes rFAAH amino acids 32-579 (encoded by residue 7 to end of SEQ ID NO:3) was amplified from the rat brain Quick-clone cDNA library (BD Biosciences Clontech) using the following primers: sense primer, δ'-GCCTCGAGACCGGGCGCCAGAAGG-S' (SEQ ID NO:12); and antisense primer, 5'-GCGAATTCTCACGATGGCTGC TTTTGAGG-3' (SEQ ID NO:13).

The resulting PCR product was digested with Xho-l-EcoR I, and subcloned into a Xho I- EcoR l-digested pTrcHis A vector to generate an NH₂-teminally His-tagged pTrcHis A- rFAAH (amino acids 32-579).

pET28a-rFAAH (amino acids 30-579 of rat FAAH is encoded by SEQ ID NO:3): The rat

FAAH construct for subcloning into the prokaryotic expression vector pET28a(+) was generated by PCR from the pTrcHis A-rFAAH (amino acids 32-579) construct using the following primers: sense primer, 5'- AAAAAAAAAAGCTAGCCGATGGACCGGGCGCCAGAAGGC-S' (SEQ ID NO:14), and antisense primer, δ'-AAAAAAAAAACTCGAGTCACGATGG CTGCTTTTGAGGGGTC-3'

(SEQ ID NO-.15). The resulting PCR product was digested with Nhe I-Xho I₁ and subcloned into a Nhe I- Xho l-digested pET28a(+) vector to generate an NH₂-terminally His-tagged pET28a- rFAAH (amino acids 30-579).

Using the methods described above, constructs were made comprising the truncated rFAAH sequences. These constructs were used to express, purify and assay the rFAAH by methods similar to those described above. The results obtained with rFAAH are summarized in Table 2. SDS PAGE (4-20% gradient) visualized by Coomassie Blue staining revealed a predominant single band corresponding to the expressed rFAAH as shown in FIGURE 1 (labeled as rFAAH)

Table 2: Optimization of rFAAH Expression

* Based on purification with a yield of 60%, 28 mg corresponds to 47 mg expressed per liter of culture (28mg x 100/60).

All publications mentioned hereinabove are hereby incorporated in their entirety by reference.

Sequence Listing Guide: SEQ ID NO. 1 Nucleotide sequence encoding amino acids 30 to 579 of wild-type homo sapien FAAH.

SEQ ID NO. 2 Nucleotide sequence encoding amino acids 30 to 579 of homo sapien FAAH, optimized for expression in E.coli.

SEQ ID NO. 3 Nucleotide sequence encoding amino acids 30-579 of full length rat FAAH

SEQ ID NO. 4 Amino acid sequence of the wild-type homo sapien FAAH. SEQ ID NO: 5 Nucleotide sequence encoding full length rat FAAH (amino acids 1-579)

SEQ ID NO: 6 The DNA sequence of the E. coli codon optimized sequence of hFAAH (amino acids 1-579) with a 5'-Xho I site, 3' stop codon, and 3'-EcoR I site.

SEQ ID NO:7 The DNA sequence of the E. coli codon optimized sequence of hFAAH (amino acids 30-105) with a 5'-Xho I site and 3'-Hind III site.

SEQ ID NO: 8-15 primers

SEQ ID NO: 16 Nucleotide sequence encoding wild-type homosapien FAAH

SEQ ID NO: 17 - Amino acid sequence expressed contiguously with amino acids 30-579 of hFAAH, as encoded by pTrcHisA-hFAAH

SEQ ID NO: 18 - Amino acid sequence expressed contiguously with amino acids 32-579 of hFAAH as encoded by pET28a-hFAAH.

SEQ ID NO: 19 - Amino acid sequence expressed contiguously with amino acids 30-579 of hFAAH, as encoded by pET-SUMO-hFAAH.

Claims

CLAlMSWhat is claimed is:

1. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: a. A nucleotide sequence set forth in SEQ ID NO:1 ; b. A nucleotide sequence that is 95 to 99% identical to a nucleotide sequence set forth in SEQ ID NO:1 ; c. A nucleotide sequence set forth in SEQ ID NO:2; d. A nucleotide sequence that is at least 70% identical to a nucleotide sequence set forth in SEQ ID NO:2; e. A nucleotide sequence encoding amino acids 30-579 of SEQ ID NO: 4; and f. A nucleotide sequence encoding amino acids 32-579 of SEQ ID NO: 4.

2. A vector comprising the nucleic acid molecule of Claim 1.

3. An isolated polypeptide encoded by the nucleic acid molecule of claim 1.

4. A vector comprising the nucleic acid molecule of Claim 1 , and further comprising a nucleic acid molecule encoding a SUMO polypeptide sequence.

5. A vector comprising the nucleic acid molecule of Claim 1 , and further comprising a promoter sequence operably linked to said nucleic acid molecule wherein said promoter is a T7 promoter.

6. A bacterial host-cell transformed with the vector according to claim 2.

7. A method for expression of a FAAH protein comprising transforming bacterial host-celis with a vector comprising a nucleic acid molecule encoding a FAAH polypeptide and inducing expression of said polypeptide wherein said induced expression yields at least 0.13 to 50 mg of FAAH polypeptide per 1 liter of cell culture.

8. The method of Claim 7, wherein said polypeptide is human FAAH and said induced expression yields at least 0.13 to 26 mg of active and soluble FAAH per

1 liter of cell culture.

9. The method of Claim 7, wherein said induction is in the presence of 50 to 300 μM of IPTG and in the presence of L-arabinose.

10. The method of Claim 7, wherein said polypeptide is rat FAAH and said induced expression yields at least 10-50 mg of active and soluble FAAH per 1 liter of cell culture.

11. A method for the expression of a FAAH protein comprising culturing the host cell of claim 6.

12. A method of producing purified FAAH polypeptide, comprising: a. Expressing a heterologous FAAH polypeptide in a host-cell, and b. Isolating the membrane fraction of said host-cells by at least one ultracentrifugation step in absence of detergent.

13. The method of Claim 12, wherein said FAAH polypeptide is encoded by a nucleic acid molecule according to claim 1.

14. A method for producing purified FAAH polypeptide, comprising: a. expressing a heterologous FAAH polypeptide in a host-cell; b. lysing said host-cells in the absence of a detergent; c. centrifuging the lysate of Step (b) at a low speed and retaining supernatant; d. ultracentrifuging said supernatant and retaining pellet; e. re-suspending said pellet in a buffer lacking detergent, f. ultracentrifuging said re-suspension and retaining pellet, g. solubilizing soluble polypeptides comprised in said pellet of Step (f) by re- suspending said pellet in a buffer comprising non-ionic detergent; h. ultracentrifuging said re-suspension of Step (g) and retaining supernatant, and i. isolating FAAH polypeptide from the supernatant of Step (h).

15. The method of Claim 14 wherein said FAAH polypeptide is fused to a histidine tag, and wherein said isolation of step i comprises column chromatography utilizing a column comprising beads charged with a cation.