U.S. patent application number 16/673775 was filed with the patent office on 2020-04-16 for method for detecting opioids, opiates, cannabinoids, or benzodiazepines in a sample with a b-glucuronidase enzyme.
The applicant listed for this patent is LA PIEDRA BIOTECNOLOG A SPA. Invention is credited to Daniel E. ALMONACID CORONADO, Manuel ROZAS ANDREU.
Application Number | 20200116719 16/673775 |
Document ID | / |
Family ID | 57119795 |
Filed Date | 2020-04-16 |
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United States Patent
Application |
20200116719 |
Kind Code |
A1 |
ROZAS ANDREU; Manuel ; et
al. |
April 16, 2020 |
METHOD FOR DETECTING OPIOIDS, OPIATES, CANNABINOIDS, OR
BENZODIAZEPINES IN A SAMPLE WITH A B-GLUCURONIDASE ENZYME
Abstract
The present invention discloses a highly efficient method for
detecting opiods, opiates, cannabinoids, or benzodiazepines present
in a sample, comprising the steps of adding to said sample an
enzyme with .beta.-glucuronidase activity originated from genus
Brachyspira or any mutant derived thereof; incubating the sample
with the enzyme; and detecting said opiods, opiates, cannabinoids,
or benzodiazepines by means of a suitable technique.
Inventors: |
ROZAS ANDREU; Manuel;
(Puerto Varas, CL) ; ALMONACID CORONADO; Daniel E.;
(Lafayette, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LA PIEDRA BIOTECNOLOG A SPA |
Puerto Varas |
|
CL |
|
|
Family ID: |
57119795 |
Appl. No.: |
16/673775 |
Filed: |
November 4, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15442607 |
Feb 24, 2017 |
|
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16673775 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/948 20130101;
G01N 2333/924 20130101; G01N 33/56916 20130101; G01N 33/9486
20130101; C12Q 1/34 20130101; G01N 30/72 20130101 |
International
Class: |
G01N 33/569 20060101
G01N033/569; C12Q 1/34 20060101 C12Q001/34; G01N 33/94 20060101
G01N033/94 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 26, 2016 |
GB |
1614546.8 |
Claims
1. A method for detecting opiods, opiates, cannabinoids, or
benzodiazepines in a sample, comprising the steps of: a) providing
the sample in which said opiods, opiates, cannabinoids, or
benzodiazepines are to be detected; b) adding to said sample an
enzyme with .beta.-glucuronidase activity originated from genus
Brachyspira or any mutant derived thereof; c) incubating the sample
with the enzyme; and d) detecting said opiods, opiates,
cannabinoids, or benzodiazepines through any suitable
technique.
2. The method of claim 1, wherein the sample is a biological
sample.
3. The method of claim 2, wherein the biological sample is selected
from the group consisting of saliva, whole blood, plasma, urine,
hair, skin, teeth, soft tissues, meconium and vitreous humor.
4. The method of claim 1, wherein the enzyme with
.beta.-glucuronidase activity originates from species Brachyspira
pilosicoli.
5. The method of claim 4, wherein the enzyme with
.beta.-glucuronidase activity is selected from the group consisting
of: a) an amino acid sequence comprising the amino acid sequence
shown in SEQ.ID.NO. 1; b) an amino acid sequence sharing at least
an 80% of identity with the amino acid sequence defined in a).
6. The method of claim 1, wherein the step of incubating the sample
with the enzyme is performed for a period between 1 and 30
minutes.
7. The method of claim 1, wherein the step of incubating the sample
with the enzyme is performed at a temperature range between 20 and
60 degrees Celsius.
8. The method of claim 1, wherein the step of incubating the sample
with the enzyme is performed at a range of pH between 4.0 and
9.0.
9. The method of claim 1, wherein the suitable technique for
detecting opiods, opiates, cannabinoids, or benzodiazepines is
selected from the group consisting of LC-MS, GC-MS, HPLC-MS,
LC-HRMS, HPLC-HRMS, LC-MS-MS, GC-MS-MS and HPLC-MS-MS.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/442,607, filed on Feb. 24, 2017, entitled "METHOD FOR
DETECTING PRODUCTS DERIVED FROM GLUCURONIDE METABOLITES WITH THE
ENZYME B-GLUCURONIDASE, AND A REAGENT COMPRISING SAID ENZYME,"
which claims priority under 35 USC .sctn. 119 to GB Patent
Application No. 1614546.8, filed on Aug. 26, 2016, entitled "METHOD
FOR DETECTING PRODUCTS DERIVED FROM GLUCURONIDE METABOLITES WITH
THE ENZYME BETA-GLUCURONIDASE, AND A REAGENT COMPRISING SAID
ENZYME," the contents of both of which are incorporated herein by
reference in their entireties.
TECHNICAL FIELD
[0002] The present invention is related with the technical field of
biotechnology, and particularly provides a method for detecting
products derived from glucuronide metabolites such as opioids,
opiates, cannabinoids, or benzodiazepines present in a sample,
using for this purpose a .beta.-glucuronidase enzyme from bacterial
origin.
BACKGROUND OF THE INVENTION
[0003] Conjugation of endogenous and exogenous compounds with
D-glucuronic acid is a metabolic pathway commonly known in animals
and humans, which is normally considered as a process for
detoxification of organisms. Through this process, glucuronide
conjugates or metabolites are produced, which are excreted through
urine or bile (Boppana V et al, J. Pharm. Sci. 1989. Vol
78(2):127-131). Compounds that are excreted in these ways are
mainly lipophilic, such as bilirubin, androgens, glucocorticoids,
among others, and xenobiotic substances such as opioids,
cannabinoids, benzodiazepines, among others.
[0004] Detection of these glucuronide metabolites in biological
fluids allows identification and quantitation of endogenous or
exogenous compounds present in the organism. Said detection can be
performed by means of chemical hydrolysis (acid hydrolysis) or by
means of enzymatic hydrolysis of said glucuronide metabolites.
Chemical hydrolysis is performed with an acid which hydrolyses most
of conjugates in a relative reduced period of time (from 60 to 90
minutes) and at a low cost. However, the acid conditions and
temperature required in this process degradate sensitive compounds,
for example, benzodiazepines. Moreover, working with acids in the
laboratory requires stricter security conditions, producing
corrosion of vulnerable equipment. For this reason, testing
laboratories for these purposes mainly employ enzymatic hydrolysis,
consisting in the use of .beta.-glucuronidases for hydrolyzation of
glucuronide metabolites in conditions more manageable than acid
hydrolysis.
[0005] Currently there exist diverse .beta.-glucuronidase enzymes
available from different origins, each one with optimum conditions
for hydrolysis of metabolites. Thus, parameters such as
concentration, pH, incubation period and temperature of reaction
vary depending on the enzyme used.
[0006] For example, there exist alternatives of enzymes obtained
from molluscs such as a .beta.-glucuronidase enzyme originated from
Patella vulgata described in document U.S. Pat. No. 4,473,640 of
Combie et al, which requires a temperature between 60 and 70
degrees Celsius, pH 5, and at least one hour to obtain only 60% of
hydrolysis of the glucuronide metabolite, and approximately three
hours as the optimum time to reach complete quantification of the
hydrolyzed metabolite. There also exist .beta.-glucuronidase
enzymes described from species Helix pomatia, Helix aspersa,
Haliotis rufescens, among others (WO 95/16050, Scheinmann et al;
Kemp et al, FAA. 2015. Report No. DOT/FAA/AM-15/6). Generally,
preparations of enzymes obtained from molluscs tend to have more
contaminant substances that may interfere with further analysis of
glucuronide metabolites.
[0007] On the other hand, there also exist alternatives to enzymes
from bacterial origin, such as .beta.-glucuronidase from
Escherichia coli. Comparative studies show that at an optimum pH
and depending on the type of glucuronide metabolite/substrate, it
is required an hour of incubation to obtain 40-80% of hydrolysis,
and between three to five hours to reach a complete hydrolysis
(Wakabashi et al, J Biol. Chem. 1961. Vol 236(4):996-1001). These
detection times are excessive for testing laboratories which
analyze drugs in biological samples, and the situation is even more
dramatic in cases such as analysis of natural opioids (codeine and
morphine) and semi-synthetic opioids (hydromorphone, oxycodone and
oxymorphone). The hydrolysis percentage of conjugated metabolites
of these opioids widely varies depending on the origin of the
enzyme used: after an incubation period of two hours of the
biological sample with .beta.-glucuronidase from Patella vulgate,
only a 21% of codeine and a 64% of morphine are recovered; an 11%
of codeine and a 35% of morphine are obtained when
.beta.-glucuronidase from Helix pomatia is used; and only a 9% of
codeine and morphine are recovered when .beta.-glucuronidase from
Escherichia coli is used (Wang et al., J Anal. Toxicol. 2006. Vol
30:570-575). For this reason, diverse researchers seek enzymes
capable of reduce the time needed to hydrolyze glucuronide
metabolites. The recently published patent application US
2016/0090582 discloses a recombinant .beta.-glucuronidase enzyme
from E. coli (IMCSzyme.TM.), which, according to the applicant,
detects the presence of drugs in biological samples in 30 minutes
or less, whose specific activity is three times higher than wild
type enzyme. However, in the examples section it is shown that 60
minutes are required to incubate the mutant enzyme with a sample
containing codeine-6-glucuronide at 55 degrees Celsius to recover
93.5% of codeine.
[0008] Therefore, new .beta.-glucuronidase enzymes are needed to
achieve quick hydrolysis of glucuronide metabolites and to obtain
high recovery percentages of their derivatives, in order to
implement a highly efficient method for detection and
quantification of the same.
SUMMARY OF INVENTION
[0009] The present invention discloses a method for detecting
opioids, opiates, cannabinoids, or benzodiazepines in a sample,
comprising the steps of: [0010] a) providing the sample in which
said opioids, opiates, cannabinoids, or benzodiazepines are to be
detected; [0011] b) adding to said sample an enzyme with
.beta.-glucuronidase activity originated from genus Brachyspira or
any mutant derived thereof; [0012] c) incubating the sample with
the enzyme; and [0013] d) detecting said opioids, opiates,
cannabinoids, or benzodiazepines through any suitable
technique.
[0014] In a preferred embodiment, the sample from which said
opioids, opiates, cannabinoids, or benzodiazepines are to be
detected, is a biological sample preferably selected from the group
consisting of saliva, whole blood, plasma, urine, hair, skin,
teeth, soft tissues, meconium, vitreous humor, water and food.
[0015] The enzyme with .beta.-glucuronidase activity of the present
invention, preferably originates from species Brachyspira
pilosicoli. In a preferred embodiment, the .beta.-glucuronidase
enzyme has the amino acid sequence shown in SEQ ID No. 1. The
invention also includes any mutant enzyme derived from SEQ ID No. 1
that maintains its .beta.-glucuronidase activity. Preferably, the
mutant enzyme with .beta.-glucuronidase activity shares at least an
80% of identity with the sequence defined in SEQ ID No. 1.
[0016] In another preferred modality of the method of the present
invention, the step of incubating the sample with the enzyme is
performed in a period of at least one minute, preferably between 2
and 120 minutes, and even more preferably between 3 and 30 minutes.
Preferably, said incubation is performed in a range of temperature
between 20 and 60 degrees Celsius, preferably between 50 and 55
degrees Celsius, and in a pH range between 4.0 and 9.0, preferably
between 6.5 and 7.5.
[0017] The detection of opioids, opiates, cannabinoids, or
benzodiazepines is performed through any suitable analytical
technique, preferably selected from the group consisting of liquid
chromatography (LC) or gas chromatography (GC), high performance
liquid chromatography (HPLC), followed by mass spectrometry (LC-MS,
or GC-MS), or high resolution mass spectrometry (LC-HRMS,
HPLC-HRMS), or tandem mass spectrometry (LC-MS-MS, GC-MS-MS,
HPLC-MS-MS) or any other suitable technique or combination of
detection methods.
BRIEF DESCRIPTION OF FIGURES
[0018] FIG. 1 shows vector pET-28a with the optimized nucleotide
sequence encoding .beta.-glucuronidase enzyme originated from B.
pilosicoli to be expressed in E. coli.
[0019] FIG. 2 shows an SDS-PAGE gel of .beta.-glucuronidase enzyme
purified with column Ni-NTA.
[0020] FIG. 3 shows recovery of codeine and morphine versus time,
using .beta.-glucuronidase from B. pilosicoli.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention relates to a method for detecting
products derived from glucuronide metabolites, preferably opioids,
opiates, cannabinoids, or benzodiazepines present in a sample,
comprising the use of a new .beta.-glucuronidase enzyme originated
from Brachyspira genus. Hydrolysis efficiency of said enzyme is
widely superior than all .beta.-glucuronidases currently known,
with a specific activity 6.6 times higher than mean specific
activity of a .beta.-glucuronidase obtained from E. coli. This
allows to detect products derived from glucuronide metabolites in
less than 15 minutes with a high recovery rate.
[0022] All the technical and scientific terms used to describe the
present invention have the same meaning understood by any person
with basic knowledge in the field under review. Nevertheless, to
define more clearly the scope of the invention, a list of
terminology used in this description is given below.
[0023] It should be understood that as used herein, the term
"glucuronide metabolite" refers to any compound conjugated with
glucuronic acid through a glycosidic bond. It is to be understood
that a glucuronide metabolite can be natural or synthetic, and
produced by chemical or enzymatic methods. The terms "conjugated
compound", "conjugated metabolite", "glucuronide compound", and
"glucuronide substrate" are indistinctly used to describe the
technical characteristics of the present invention.
[0024] The term "products derived from glucuronide metabolites" or
"recovered analytes" should be understood as the molecule or
original compound that is produced after hydrolysis or glycosidic
bond cleavage of a glucuronide metabolite. These derived products
or original compounds can be natural or synthetic, and produced by
chemical or enzymatic methods.
[0025] The term "enzyme" should be understood as a sequence of
amino acids which form a functional protein of interest for the
present invention. It is to be understood that the present
invention is not related with enzymes in their natural state, but
it relates to proteins in an isolated state, purified or partially
purified, or recombinant, obtained by any method of genetic
engineering known in the state of the art. Likewise, it is to be
understood that the term "mutant" or "mutant enzyme" refers to a
modified protein derived from the original amino acid sequence
maintaining its .beta.-glucuronidase enzymatic activity; and
sharing at least 70%, preferably 80% and more preferably 90% of
identity with the original sequence.
[0026] It is to be understood that the term "identity" among amino
acid sequences refers to the percent of identical amino acids that
the compared sequences share among them, in a particular sequence
alignment window. The percentage of identity can be calculated
using a sequence comparison algorithm or by manual alignment
together with visual inspection. For example, sequences and
percentages of identity can be obtained using computer resources
available on internet such as BLAST
(http://blast.ncbi.nlm.nih.gov/) or FastDB computer programs.
[0027] The term "Unit (U)" should be understood as the unit used to
measure the specific activity of an enzyme. One unit will produce
1.0 .mu.g of phenolphthalein from phenolphthalein glucuronide (PPG)
metabolite per hour at 37 degrees Celsius at pH 6.8.
[0028] The term "vehicle" should be understood as any substance
that is added to an active principle, in this case an enzyme, which
is useful as a support and allows to dilute or improve stability or
durability of the main compound.
[0029] As described before, an object of the present invention
relates to a method for detection of glucuronide metabolites in a
sample, which comprises the steps of adding an enzyme with
.beta.-glucuronidase activity from genus Brachyspira or any variant
or mutant derived thereof to the sample in which a product derived
from a glucuronide metabolite is to be detected, incubating the
sample with the enzyme for a determined period of time; and
detecting the product derived from said glucuronide metabolite
through any suitable technique.
[0030] This method is superior than others described in the state
of the art, since it includes the use of the new enzyme with
.beta.-glucuronidase activity originated from genus Brachyspira,
which has an improved affinity for glucuronide metabolites, a
specific activity 6.6 times higher than .beta.-glucuronidase
originated from E. coli, and therefore it is capable of hydrolyse
glucuronide metabolites to be detected in a period of time of at
least one minute, preferably between three and 30 minutes. This
enzyme is preferably originated from species Brachyspira
pilosicoli, whose amino acid sequence is shown in SEQ ID No. 1, but
it can be obtained from any species of genus Brachyspira.
Additionally, in the method of the present invention, any
derivative or mutant of this protein can be used, provided that it
maintains its .beta.-glucuronidase activity. Preferably, the
derived or mutant enzyme with .beta.-glucuronidase activity shares
at least a 70% of sequence identity, more preferably an 80% of
identity, and even more preferably a 90% of identity with the
sequence defined in SEQ ID No. 1.
[0031] The .beta.-glucuronidase enzyme originated from Brachyspira
can hydrolyse any glucuronide metabolite, whether from natural,
semi-synthetic or synthetic origin. Some examples of these
metabolites, without limitation, are: morphine-3-glucuronide,
morphine-6-glucuronide, codeine-6-glucuronide,
oximorphone-glucuronide, hydromorphone-glucuronide,
norbuprenorphine-glucuronide, buprenorphine-glucuronide,
oxazepam-glucuronide, temazepam-glucuronide, lorazepam-glucuronide,
alprazolam-glucuronide, midazolam-glucuronide,
nordiazepam-glucuronide, psilocin-glucuronide,
carboxy-tetrahydrocannabinol-glucuronide,
tetrahydrocannabinol-glucuronide, tetrahydrocannabinolic
acid-glucuronide, naloxone-3-glucuronide, tapentadol-glucuronide,
tricyclic antidepressant-glucuronide, cannabidiol-glucuronide,
among others. It is to be understood for the scope of the present
invention that the .beta.-glucuronidase enzyme originated from
Brachyspira can hydrolise any glucuronide metabolite and it is not
limited to the examples previously mentioned.
[0032] Therefore, this method allows to detect any product derived
from glucuronide metabolites, whether from natural, semi-synthetic
or synthetic origin. For example, the method can be used to analyze
drugs, toxic compounds derived from fungi, androgens, estrogens,
mineralocorticoids, glucocorticoids, fatty acid derivatives,
retinoids, among others.
[0033] In a preferred embodiment, products derived from glucuronide
metabolites are selected from the group consisting of opioids,
opiates, cannabinoids, benzodiazepines or any other derived
thereof. Preferably, the products derived from glucuronide
metabolites are selected from the group consisting of morphine,
codeine, oximorphone, hydromorphone, norbuprenorphine,
buprenorphine, oxazepam, temazepam, lorazepam, alprazolam,
midazolam, nordiazepam, psilocin,
11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH),
tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCa),
naloxone, tapentadol, tricyclic antidepressant, cannabidiol, among
others, without being limited to the examples described herein.
[0034] In a preferred embodiment of the present invention, the
method allows to detect any product derived from glucuronide
metabolites in any type of sample, which is preferably selected
from the group consisting of saliva, whole blood, plasma, urine,
hair, skin, teeth, soft tissues, water, food, meconium and vitreous
humor, without restriction to these types of samples. The following
biological samples can also be used: bile, earwax, phlegm, vomit,
aqueous humour, tears, amniotic fluid, vaginal secretion, semen,
pre-ejaculate fluid, mucus, sebum, sweat, excrement, or any other
suitable sample without limitation to the animal fluids previously
mentioned. Other suitable samples originate from a non-animal
source, such as food, water or chemical solutions from
laboratories.
[0035] In the method of the present invention, .beta.-glucuronidase
enzyme originated from genus Brachyspira is added to the sample and
the mixture is incubated for a determined period of time, in
suitable conditions. In a preferred embodiment, incubation period
of sample with the enzyme is of at least one minute, that is to
say, only one minute of reaction is enough to detect the presence
of a product derived of a glucuronide metabolite in a sample.
However, incubation period can be extended to 16 hours or even
more, provided that time is not an impediment to the technician
performing the method.
[0036] More preferably, incubation period is between 2 and 120
minutes, and even more preferably, incubation period is between 3
and 30 minutes, since obtaining the results efficiently and in the
shortest time is commonly desired. Additionally, adequate
incubation conditions refer to ideal conditions of temperature and
pH in order to allow the reaction to be produced in the time
periods mentioned. Regarding temperature, incubation is performed
preferably in a range between 20 and 60 degrees Celsius, and even
more preferably in a range of 50 to 55 degrees Celsius. Regarding
pH, incubation is performed in a range between 4.0 and 9.0 and
preferably between 6.5 and 7.5. As part of the elements routinely
used in these assays, incubation is performed in presence of buffer
solutions, standards of products derived from glucuronide
metabolites, polar or apolar solvents, among others.
[0037] After incubating the .beta.-glucuronidase enzyme with the
sample and before separating the products derived from glucuronide
metabolites through separation columns, an extraction step of said
metabolites can be optionally included to increase sensitivity of
analytical techniques. This extraction step can be performed by
means of liquid-liquid extraction (LLE), solid-liquid extraction
(SLE), extraction in solid phase (SPE), or any other suitable
technique. However, the step of extracting the products derived
from glucuronide metabolites adds a laborious step to the
analytical method and increases operational costs due to the use of
solvents and other materials.
[0038] To the method of the present invention, the latter step is
optional due to the availability of highly selective analytical
techniques that allow direct analysis by means of the technique
named "dilute and shoot", which consist of diluting the sample with
an adequate solution and injecting it directly into a separation
column. Moreover, the method does not require elimination of the
enzyme since the .beta.-glucuronidase of the present invention is
highly purified and has a high reactivity; and therefore, a small
volume of the enzyme is required to react with glucuronide
metabolites. In a preferred embodiment the volume range of enzyme:
liquid sample used, for example, urine, is from 1:1 to 1:14.
Preferably, optimum concentration is between 0.02 and 0.25 mg of
enzyme per mL of total reaction. In this manner, the volume of
enzyme used does not affect or damage the separation column in case
it is directly injected to it. The adequate solvent can be for
example, water or a mixture of water with methanol, or any mixture
of solvents that can be used to dilute the sample.
[0039] Finally, detection of products derived from glucuronide
metabolites is performed through the use of separation columns and
analytical techniques which are selected from the group consisting
of: gas chromatography (GC), liquid chromatography (LC) or high
performance liquid chromatography (HPLC), solid phase
microextraction (SPME), with mass spectrometry detectors (GC-MS;
LC-MS; HPLC-MS; SPME-MS), high resolution mass spectrometry
(GC-HRMS; LC-HRMS; HPLC-HRMS), tandem mass spectrometry (GC-MS/MS;
LC-MS/MS; SPME-GC-MS/MS), time of flight mass spectrometry
(LC-TOF-MS), ultraviolet-visible spectrophotometry (GC-UV/Vis;
LC-UV/Vis; SPME-LC-UV/Vis), flame ionization detection (GC-FID;
LC-FID; SPME-GC-FID), photodiode array detection, (LC-DAD), or any
other suitable detection method, without limiting to the examples
previously mentioned.
[0040] A second object of the present invention is a reagent for
detection of products derived from glucuronide metabolites in a
sample, which comprises an enzyme with .beta.-glucuronidase
activity originated from genus Brachyspira or any derived enzyme or
mutant thereof; which is included in a suitable vehicle.
[0041] Preferably, the reagent contains an enzyme with
.beta.-glucuronidase activity originated from species Brachyspira
pilosicoli, or any derived enzyme or mutant thereof. Said enzyme
has an amino acid sequence shown in SEQ ID No. 1, but it can be any
enzyme mutant from said sequence and maintaining
.beta.-glucuronidase activity. Preferably, the derived or mutant
enzyme with .beta.-glucuronidase activity shares at least a 70% or
80% of identity with the sequence defined in SEQ ID No. 1, more
preferably a 90% of identity with the sequence defined in SEQ ID
No. 1.
[0042] In a preferred embodiment, the reagent contains between 0.1
and 2 mg/mL of enzyme, in which the same has a .beta.-glucuronidase
activity from 200,000 U/mL to 800.000 U/mL. In addition to
.beta.-glucuronidase enzyme, the reagent includes an appropriate
vehicle that varies depending on the product format and on the
function that the same performs. For example, the reagent including
the enzyme can be in a liquid format, which could require only
water as a vehicle.
[0043] To improve the stability of the enzyme, salts can be added
(at concentrations .mu.M or mM), or ethylenediamine tetraacetic
acid (EDTA), Tris, dithiothreitol (DTT),
hydroxyethylpiperazin-ylethanesulfonic acid (HEPES), citric acid,
among others, or a combination thereof. Preferably, salts are
selected from the group consisting of sodium phosphate, potassium
phosphate, sodium carbonate, sodium acetate, sodium citrate, sodium
chloride and potassium chloride, without limitation to the salts
previously described. In case that the reagent is frozen, the
vehicle to be used can include glycerol, preferably at a
concentration of 50%. On the other hand, if the reagent format is
powder, which can be produced by means of lyophilisation, spray
drying or any other pulverization method; vehicles able to protect
the enzyme from drying process are required such as sugars, amino
acids and some salts, which are present at a concentration range
between 0.001 to 2% based on liquid previous to the drying process.
In this case, sugars are preferably selected from the group
consisting of sorbitol, trehalose, sucrose, glucose, lactose,
mannitol and raffinose, without limitation to these described
examples. Therefore, the vehicle of the present invention fulfils
the purpose of being a diluting, preservating or stabilizing medium
for the enzyme.
[0044] The following examples are considered to illustrate the
invention and its preferred embodiments, but they must not be
considered under any circumstance as a restriction to the scope of
the invention, which is defined by the claims attached hereto.
EXAMPLES
Example 1: Synthesis and Expression of .beta.-glucuronidase Enzyme
from B. pilosicoli
[0045] To express the .beta.-glucuronidase enzyme, nucleotide
sequence from said protein was used, originated from Brachyspira
pilosicoli strain B2904 described in Genbank (access code
CP003490), which is shown in SEC.ID.NO.2, and codon optimization
was performed using the algorithm OptimumGene.TM. (GenScript) to
efficiently express said sequence in E. coli. To do this, codon
adaptation index (CAI), understood as distribution of frequency of
codon usage through the sequence, was adjusted from 0.66 to 0.88,
in which a CAI value of 1.0 is considered to be perfect in the
desired expression organism; and a CAI value greater than 0.8 is
regarded as good in terms of high gene expression levels.
Guanine-cytosine content was also optimized to extend mRNA
half-life and stem-loop structures were eliminated, since they
disrupt ribosome binding and mRNA stability. Optimized nucleotide
sequence obtained is shown in SEC.ID.NO.3. Amino acid sequence of
the enzyme is shown in SEC.ID.NO.1.
[0046] The optimized nucleotide sequence of the enzyme was cloned
into vector pET-28a, thus vector pET-28a-glucuronidase was
obtained, which is shown in FIG. 1. To express .beta.-glucuronidase
enzyme from B. pilosicoli in E. coli, the following protocol was
carried out: 5 .mu.L of competent E. coli strain BL21 cells were
incubated in ice with 10 ng of pET-28a-glucuronidase vector during
20 minutes. Then, bacterial cells were submitted to heatshock at 37
degrees Celsius during 3 minutes. Bacterial cells were then put in
ice for 30 seconds and later cells were recovered with 250 .mu.L of
LB medium at 37 degrees Celsius and plated in LB-agar with 50 mg/L
of kanamycin, and colonies were grown. Finally, a
.beta.-glucuronidase activity test was carried out on plates with
IPTG (isopropyl-.beta.-thiogalactoside) and X-Gal
(5-bromo-4-chloro-3 indolyl-beta-D-thiogalactopyranoside).
[0047] In order to purify the enzyme, E. coli BL21 transformed
cells were grown in 500 mL of LB medium supplemented with kanamycin
(50 mg/L), until obtention of an optical density of 0.3, measured
using a spectrophotometer at 600 nm (DO.sub.600).
[0048] Then, IPTG at a concentration of 1 mM was added to the
culture and then incubated at 37 degrees Celsius during 6 hours.
Cells were subjected to sonication during 3 minutes with an
amplitude of 60 (cycles of 15 seconds in ice). Later, the enzyme
was purified by means of a system for protein purification in
columns with Ni-NTA (Nickel-Nitriloacetic acid) resins with elution
of 2 mL of Tris-HCl 50 mM, pH 5.6 with imidazole 100 mM pH 5.9 or
with imidazole 500 mM pH 4.5.
[0049] FIG. 2 shows an SDS-PAGE gel of purified proteins in column
Ni-NTA. Each well contained proteins at a concentration of 15
.mu.g/mL, except in crude extract (E. coli pET28a-glucuronidase
without purification in the column), in which 5 .mu.L were loaded.
The acronym Std corresponds to molecular weight standard in
kilodaltons (kD), Crude ex corresponds to crude extract, Glu E1
corresponds to elution with imidazole 100 mM pH 5.9 and Glu E2
corresponds to elution with imidazole 500 mM pH 4.5.
Example 2. Incubation Time of a Sample with .beta.-glucuronidase
from B. pilosicoli
[0050] The required time to hydrolyze morphine-3-glucuronide and
codeine-6-glucuronide with the enzyme .beta.-glucuronidase in
excess (named BGTurbo.TM.) from B. pilosicoli was evaluated with
the following protocol: 10 .mu.L of glucuronide metabolites at a
concentration of 100 .mu.g/mL, free standards (free codeine and
morphine calibrators at concentrations of 312.5, 625, 1,000, 1,250
and 1,875 ng/mL), and internal standards (40 .mu.L of deuterated
free drug at a concentration of 1 .mu.g/mL to each sample) were
added to a sample of 0.4 mL of blank urine and then submitted to
vortex agitation. Then, 360 .mu.L of sodium phosphate (pH 7; 140
mM) solution was added to each sample. Then, 240 .mu.L of
.beta.-glucuronidase (BGTurbo.TM.) were added to each sample and
then agitated by vortex. Samples were incubated at 55 degrees
Celsius in an oven during 0, 10, 20, 30, 40, 50 and 60 minutes. A
total of 21 samples were used: 7 points of measure at different
times, each one in triplicate. Calibrators were incubated for 30
minutes. The samples incubated at different times were removed from
oven and the reaction was stopped by adding 700 .mu.L of sodium
phosphate buffer solution 1.5 M and agitated by vortex. Then, pH
8-9 was verified with pH paper, and 2 mL of chloroform: isopropanol
50:50 were added to samples, and they were agitated by vortex for 2
minutes. Aqueous phase was discarded, and organic phase was
transferred to 12.times.75 mm assay tubes and then evaporated under
nitrogen gas (in a water bath at 50 degrees Celsius) until
approximately 1 mL. Samples were then agitated by vortex previous
to evaporation until dryness. Samples were reconstituted in 1 mL of
HCl 0.5 N in methanol for LC-MS/MS analysis.
[0051] Calculation of concentration of equivalents was performed as
follows:
Morphine:
[0052] Molecular weight of morphine: 285.34 g/mol
[0053] Molecular weight of morphine-3-glucuronide: 461.462
g/mol
[0054] Proportion of molecular weight of
morphine/morphine-3-glucuronide:
[0055] Morphine equivalents:
0.6183 .times. 2 , 500 ng mL Morphine - 3 - glucuronide = 1 , 545.8
ng mL Morphine ##EQU00001##
Codeine:
[0056] Molecular weight of codeine: 299.364 g/mol
[0057] Molecular weight of codeine-6-glucuronide: 475.49 g/mol
[0058] Proportion of molecular weight of
codeine/codeine-6-glucuronide:
Codeine equivalents:
0.62959 .times. 2 , 500 ng mL Codeine - 6 - glucuronide = 1 , 574.0
ng mL Codeine ##EQU00002##
[0059] Table 1 and FIG. 3 show the results obtained. Hydrolysis of
100% of codeine-6-glucuronide was obtained in less than 10 minutes.
In the case of morphin, approximately 65% of hydrolysis was
obtained within 10 minutes, but these values can be improved by
optimizing parameters of temperature, pH or the method to stop
reaction. Due to the use of internal controls for recovery of
products derived from glucuronide metabolites, values have a
standard deviation up to 20%.
TABLE-US-00001 TABLE 1 Tabulated results of FIG. 3. Time Codeine %
Yield Morphine % Yield (minutes) (ng/mL) Codeine (ng/mL) Morphine 0
30.0 1.9 195.8 12.7 10 1826.9 116.1 997.4 64.5 20 1867.5 118.6
969.2 62.7 30 1694.3 107.6 871.2 56.4 40 1821.0 115.7 811.5 52.5 50
1844.3 117.2 847.4 54.8 60 1819.5 115.6 823.4 53.3
Example 3: Calculation of Specific Activity of .beta.-glucuronidase
from B. pilosicoli
[0060] Specific activity of the .beta.-glucuronidase enzyme from B.
pilosicoli (BGTurbo.TM.) was calculated and compared with a mean
value of three calculations of the specific activity of
.beta.-glucuronidase from E. coli, using the following protocol:
350 .mu.L of sodium phosphate buffer solution (NaH.sub.2PO.sub.4)
0.1 M, pH 6.8 were mixed with 350 .mu.L of phenolphthalein
.beta.-D-glucuronide sodium salt (0.64 mg/mL) and the mixture was
incubated at 37 degrees Celsius. Then, 50 .mu.L of solution
containing the enzyme were added to the mixture and incubated at 37
degrees Celsius during 30 minutes. After the first 15 minutes the
mixture was gently agitated and stopped at 30 minutes from the
beginning of the reaction with 2.5 mL of a glycine solution 0.2 M,
pH 10.4. Absorbance was read at 540 nm and the amount of enzyme
used was calculated (U/mL) when adjusting the values in a
calibration curve. Protein concentration was measured using
Bradford method, as widely known in literature. As shown in Table
2, results indicate that BGTurbo.TM. enzyme has a specific activity
6.6 times higher than mean specific activity of
.beta.-glucuronidase from E. coli.
TABLE-US-00002 TABLE 2 Enzymatic activity of .beta.-glucuronidase
from B. pilosicoli. Activity Proteins Specific Activity Enzyme
(U/mL) (mg/mL) (U/mg) BGTurbo .TM. 427,805 2.08 205,676 E. coli 1
68,779 2.28 30,166 E. coli 2 102,710 3.17 32,401 E. coli 3 85,084
2.89 29,441 E. coli mean value 30,669
Example 4. Comparison of .beta.-glucuronidase from B. pilosicoli
with .beta.-glucuronidases from Different Sources
[0061] To test the efficiency of .beta.-glucuronidase from B.
pilosicoli (BGTurbo.TM.), this enzyme was compared with two
.beta.-glucuronidases currently available in the market: DR2102
(Campbell Science) and BG100.TM. (KURA Biotec). To do this, 300 ng
of each of the glucuronide metabolites of buprenorphine, codeine,
hydromorphone, lorazepam, morphine, oxymorphone and
11-nor-9-carboxy-tetrahydrocannnabinol (THC-COON) were added to a
sample of blank urine, therefore obtaining spiked urine. Each
enzyme was used under the conditions proposed by each manufacturer.
Hydrolysis of 50 .mu.L of urine was performed, diluting it with 250
.mu.L of buffer solution at pH 6.8, 20 .mu.L of internal standards
(ISTD) and 10 .mu.L of DR2102 or BG100 enzymes, or 20 .mu.L of
BGTurbo.TM.. Incubation conditions are shown in Table 3.
TABLE-US-00003 TABLE 3 Conditions used for each enzyme. Conditions
DR2102 BG100 .TM. BGTurbo .TM. Urine (.mu.L) 50 50 50 ISTD (.mu.L)
20 20 20 Buffer (.mu.L) 25 25 25 Enzyme (.mu.L) 10 10 20
Temperature (.degree. C.) 62 68 50 Time (minutes) 60 30 15
[0062] After hydrolysis, samples were centrifuged at 24,000 g,
diluted with 450 .mu.L of a solution comprising 95% water and 5%
methanol, and charged to an HPLC column (Phenomenex). Protein
amount in samples was measured before and after centrifugation at
24,000 g using a NanoDrop spectrophotometer (Thermo Scientific). As
a result, the reactions performed were carried out with 1.787 mg/mL
of DR2102, 0.554 mg/mL of BG100.TM. and 0.103 mg/mL of
BGTurbo.TM..
[0063] Recovery yields of products derived from glucuronide
metabolites are shown in Table 4. As shown, mean recovery of
derived products was improved in a 15% when hydrolysis was
performed with BGTurbo.TM. in only 15 minutes and using 70% and 94%
less of protein amount in comparison to the other two enzymes. It
is interesting to mention that .beta.-glucuronidase enzyme from B.
pilosicoli is more reactive with codeine-6-glucuronide than the
other enzymes.
TABLE-US-00004 TABLE 4 Results of analites recovered from
hydrolysis of glucuronide metabolites with three
.beta.-glucuronidases enzymes from different origins. DR2102 BG100
.TM. BGTurbo .TM. 60 minutes 30 minutes 15 minutes Recovered
Analyte (ng/mL) (ng/mL) (ng/mL) Buprenorphine 223.1 271.1 224.4
Codeine 159.4 173.8 243.2 Hydromorphone 176.8 196.0 196.2 Lorazepam
214.9 202.5 185.9 Morphine 174.5 190.9 198.1 Oximorphone 155.8
189.0 169.4 THC-COOH 75.5 146.0 140.6 Protein in sample 1.787
(mg/mL) 0.554 (mg/mL) 0.103 (mg/mL) pre-centrifugation Protein in
sample 0.725 (mg/mL) 0.280 (mg/mL) 0.085 (mg/mL)
post-centrifugation and injected on HPLC column
Example 5. Comparison of .beta.-glucuronidase from B. pilosicoli
with other .beta.-glucuronidases from Bacterial Origin
[0064] In order to test enzyme speed of .beta.-glucuronidase from
B. pilosicoli (BGTurbo.TM.) in comparison with
.beta.-glucuronidases from bacterial origin, BGTurbo.TM. was
compared with EBG enzyme (recombinant .beta.-glucuronidase from E.
coli, KURA Biotec) and with IMCSzyme.TM. (Integrated
Micro-Chromatography Systems) at different incubation periods and
concentration. The method previously described was used but
analyzing only hydrolysis of codeine-6-glucuronide and
morphine-3-glucuronide with the conditions described in Table
5.
TABLE-US-00005 TABLE 5 Conditions used for each enzyme. BGTurbo
.TM. EBG IMCSzyme .TM. Control Conditions A B C D F -- E Buffer
(.mu.L) 360 360 360 360 360 360 360 Water (.mu.L) 0 120 180 210 180
180 120 Spiked urine (.mu.L) 400 400 400 400 400 400 400 Enzyme
(.mu.L) 240 120 60 30 60 60 0 Internal Standard (.mu.L) 40 40 40 40
40 40 40 Storage buffer control (.mu.L) 0 0 0 0 0 0 120 Total
Volume (.mu.L) 1,040 1,040 1,040 1,040 1,040 1,040 1,040 Incubation
Time (minutes) 10 10 10 10 0, 10, 20 0, 10, 20 10 pH 7.0 7.0 7.0
7.0 7.0 7.0 7.0 Temperature (.degree. C.) 55 55 55 55 55 55 55
[0065] Each condition shown in Table 5 was assayed in duplicate,
and each analysis was measured in triplicate. Calculation of
concentration of equivalents was performed as described in Example
2.
[0066] Table 6 shows the results obtained from analysis of codeine,
and Table 7 shows results obtained from analysis of morphine. As
can be noticed, codeine-6-glucuronide was hydrolyzed in a mean
value of 109.59% within 10 minutes, using 240 .mu.L of BGTurbo.TM..
The value mentioned is greater than 100% because standards of
glucuronide metabolites used in the analysis are not completely
pure and present a fraction of the original compound that is added
to the final result. On the other hand, when comparing hydrolysis
yield of codeine-6-glucuronide and morphine-3-glucuronide using the
same volume of the enzymes (60 .mu.L), it is shown that BGTurbo.TM.
is better than the other EBG and IMCSzyme.TM. enzymes within 10
minutes. Even after an incubation period of 20 minutes of the
samples with EBG and IMCSzyme.TM., these enzymes could not produce
the same hydrolysis yield that was obtained with BGTurbo.TM. within
10 minutes.
TABLE-US-00006 TABLE 6 Results of codeine analysis. Condition/
Codeine Codeine Average of Assay Sample (ng/mL) % Yield Assay Yield
(%) A/1 BGTurbo .TM. (10 min, 240 .mu.L) 1,740 110.55 109.59 A/2
BGTurbo .TM. (10 min, 240 .mu.L) 1,710 108.64 B/1 BGTurbo .TM. (10
min, 120 .mu.L) 1,270 80.69 78.14 B/2 BGTurbo .TM. (10 min, 120
.mu.L) 1,190 75.60 C/1 BGTurbo .TM. (10 min, 60 .mu.L) 879 55.84
54.19 C/2 BGTurbo .TM. (10 min, 60 .mu.L) 827 52.54 D/1 BGTurbo
.TM. (10 min, 30 .mu.L) 230 14.61 22.20 D/2 BGTurbo .TM. (10 min,
30 .mu.L) 469 29.80 F/1 EBG (0 min, 60 .mu.L) No Peak 0.00 0.00 F/2
EBG (0 min, 60 .mu.L) No Peak 0.00 F/1 EBG (10 min, 60 .mu.L) 154
9.78 10.48 F/2 EBG (10 min, 60 .mu.L) 176 11.18 F/1 EBG (20 min, 60
.mu.L) 396 25.16 27.10 F/2 EBG (20 min, 60 .mu.L) 457 29.03 1
IMCSzyme .TM. (0 min, 60 .mu.L) No Peak 0.00 0.00 2 IMCSzyme .TM.
(0 min, 60 .mu.L) No Peak 0.00 1 IMCSzyme .TM. (10 min, 60 .mu.L)
189 12.01 11.85 2 IMCSzyme .TM. (10 min, 60 .mu.L) 184 11.69 1
IMCSzyme .TM. (20 min, 60 .mu.L) 395 25.10 27.26 2 IMCSzyme .TM.
(20 min, 60 .mu.L) 463 29.42 E/1 Control (10 min) No Peak 0.00 0.00
E/2 Control (10 min) No Peak 0.00
TABLE-US-00007 TABLE 7 Results of the morphine analysis. Condition/
Morphine Morphine Average of Assay Sample (ng/mL) % Yield Assay
Yield (%) A/1 BGTurbo .TM. (10 min, 240 .mu.L) 796 51.49 51.49 A/2
BGTurbo .TM. (10 min, 240 .mu.L) 796 51.49 B/1 BGTurbo .TM. (10
min, 120 .mu.L) 717 46.38 46.90 B/2 BGTurbo .TM. (10 min, 120
.mu.L) 733 47.42 C/1 BGTurbo .TM. (10 min, 60 .mu.L) 687 44.44
43.70 C/2 BGTurbo .TM. (10 min, 60 .mu.L) 664 42.96 D/1 BGTurbo
.TM. (10 min, 30 .mu.L) 498 32.22 38.46 D/2 BGTurbo .TM. (10 min,
30 .mu.L) 691 44.70 F/1 EBG (0 min, 60 .mu.L) 179 11.58 12.71 F/2
EBG (0 min, 60 .mu.L) 214 13.84 F/1 EBG (10 min, 60 .mu.L) 609
39.40 39.62 F/2 EBG (10 min, 60 .mu.L) 616 39.85 F/1 EBG (20 min,
60 .mu.L) 629 40.69 43.47 F/2 EBG (20 min, 60 .mu.L) 715 46.25 1
IMCSzyme .TM. (0 min, 60 .mu.L) 142 9.19 10.19 2 IMCSzyme .TM. (0
min, 60 .mu.L) 173 11.19 1 IMCSzyme .TM. (10 min, 60 .mu.L) 603
39.01 39.20 2 IMCSzyme .TM. (10 min, 60 .mu.L) 609 39.40 1 IMCSzyme
.TM. (20 min, 60 .mu.L) 662 42.83 43.41 2 IMCSzyme .TM. (20 min, 60
.mu.L) 680 43.99 E/1 Control (10 min) No Peak 0.00 0.00 E/2 Control
(10 min) No Peak 0.00
Sequence CWU 1
1
31603PRTBrachyspira pilosicoli 1Met Val Asn Ser Met Leu Tyr Pro Arg
Glu Ser Arg Thr Arg Arg Val1 5 10 15Val Asp Ile Ser Gly Met Trp Glu
Phe Lys Ile Asp Ile Asn Asn Glu 20 25 30Gly Arg Asn Ser Gly Tyr Ala
Asn Gly Leu Lys Asp Thr Thr Phe Ile 35 40 45Pro Val Pro Ser Ser Phe
Asn Asp Leu Phe Thr Asp Lys Asn Ile Arg 50 55 60Glu His Ala Gly Asp
Val Trp Tyr Glu Thr Ser Phe Tyr Leu Pro Leu65 70 75 80Glu Trp Lys
Asp Lys Asp Val Asn Val Arg Phe Gly Cys Ala Thr His 85 90 95Glu Ala
Thr Val Tyr Ile Asn Gly Lys Glu Val Cys Thr His Val Gly 100 105
110Gly Phe Met Pro Phe Asn Ala Pro Val Asn Glu Ala Gly Ile Phe Gly
115 120 125Glu Lys Asn Lys Leu Val Val Val Val Asn Asn Glu Leu Ser
Asn Thr 130 135 140Thr Ile Pro Cys Gly His Thr Glu Thr Lys Pro Ser
Gly Lys Lys Tyr145 150 155 160Ile Lys Pro Ser Phe Asp Phe Phe Asn
Tyr Ala Gly Leu Asn Arg Pro 165 170 175Val Lys Ile Thr Val Thr Asn
Lys Glu Tyr Ile His Asp Ile Asp Ile 180 185 190Leu Ser Asp Val Asn
Gly Ser Asp Gly Ile Val Asn Tyr Glu Val His 195 200 205Thr Thr Gly
Glu Asn Lys Val Tyr Ile Lys Ile Asn Asp Glu Glu Gly 210 215 220Lys
Glu Val Ala Ser Cys Glu Gly Lys Ser Gly Lys Ile Val Ile Lys225 230
235 240Asp Ala Lys Leu Trp Asn Pro Lys Ala Ala Tyr Leu Tyr Lys Phe
Ile 245 250 255Ala Cys Ile Lys Asn Gly Asp Glu Leu Ile Asp Glu Tyr
Tyr Leu Asp 260 265 270Phe Gly Ile Arg Thr Val Lys Val Glu Gly Thr
Lys Phe Leu Ile Asn 275 280 285Gly Lys Pro Phe Tyr Phe Thr Gly Phe
Gly Lys His Glu Asp Ser Glu 290 295 300Ile Ala Gly Arg Gly Tyr Asn
Pro Pro Val Ile Lys Arg Asp Phe Glu305 310 315 320Leu Ile Lys Trp
Val Gly Ala Asn Ser Phe Arg Thr Ser His Tyr Pro 325 330 335Tyr Ser
Glu Glu Ile Met Gln Ala Ala Asp Arg Glu Gly Ile Val Ile 340 345
350Ile Asp Glu Val Ala Ala Val Gly Met Phe Asp Val Gly Ser Val Leu
355 360 365Asn Pro Ser Ala Ser Lys Thr Asp Tyr Phe Ser Leu Asp Glu
Val His 370 375 380Ser Lys Thr Lys Glu Val His Lys Lys Ala Val Glu
Glu Leu Ile Lys385 390 395 400Arg Asp Lys Asn His Pro Ser Val Val
Met Trp Ser Leu Phe Asn Glu 405 410 415Pro Asp Thr Ser Lys Asp Glu
Ala Val Pro Tyr Phe Glu Asp Ile Phe 420 425 430Asn Phe Ala Lys Ser
Gln Asp Lys Gln Asn Leu Pro Lys Thr Phe Ala 435 440 445Ala Ile Gln
Ala Ser Ser Pro Gly Lys Cys Lys Cys Met His Leu Cys 450 455 460Asp
Val Ile Thr Leu Asn Arg Tyr Tyr Gly Trp Tyr Phe Leu Gly Gly465 470
475 480Tyr Glu Ile Asp Met Ser Glu Glu Lys Phe Arg Glu Glu Met Asn
Leu 485 490 495Tyr Ser Asn Met Asn Lys Pro Val Met Phe Thr Glu Tyr
Gly Ala Asp 500 505 510Thr Tyr Ala Gly Val His Lys Leu Pro Ser Val
Met Trp Ser Glu Glu 515 520 525Tyr Gln Cys Glu Tyr Tyr Glu Met Asn
Phe Lys Val Phe Asp Ser Tyr 530 535 540Asp Phe Ile Val Gly Glu Gln
Leu Trp Asn Phe Ala Asp Phe Gln Thr545 550 555 560Thr Glu Gly Ile
Phe Arg Val Asp Gly Asn Lys Lys Gly Ile Phe Thr 565 570 575Arg Asn
Arg Gln Pro Lys Ala Val Ala His Leu Ile Arg Ser Arg Trp 580 585
590Asn Lys Leu Pro Leu Asp Tyr Lys Ser Lys Lys 595
60021809DNABrachyspira pilosicoli 2atggttaatt ctatgttata tcctagagag
agtaggacaa gaagagtagt agatatatct 60ggtatgtggg agtttaaaat tgatataaat
aatgaaggca gaaatagcgg atatgctaat 120gggcttaaag atactacatt
tatacctgtt ccatctagtt ttaatgattt atttacagat 180aaaaatataa
gagaacatgc tggtgatgta tggtatgaaa catcatttta tcttcctttg
240gaatggaaag acaaagatgt taatgtaaga tttggttgtg caactcatga
agctactgta 300tatattaatg gaaaagaagt atgtacacat gtaggtggat
ttatgccttt caatgctcct 360gtaaatgaag caggaatatt tggagaaaaa
aataaattag ttgtagttgt taataatgaa 420cttagcaata ctactatacc
ttgtggtcat acagaaacta aaccttcagg caaaaaatat 480attaaacctt
cttttgactt ttttaattat gcgggattaa acagacctgt aaaaattact
540gttacaaata aggaatatat acatgatata gatatattat cagatgtaaa
cggcagtgat 600ggaattgtaa attatgaagt acatactaca ggcgaaaaca
aagtatatat aaaaataaac 660gatgaagagg gaaaagaagt agcaagttgt
gaaggtaaaa gcggaaaaat agttattaaa 720gatgctaaat tatggaatcc
aaaggctgct tatttgtata agtttattgc atgcataaaa 780aatggcgatg
aactcataga tgaatattat ttagactttg gtataagaac tgttaaagtt
840gaaggaacaa aatttttaat taatggaaaa cctttctact ttacaggatt
tggtaaacat 900gaagacagtg aaatagcagg taggggttat aatcctcctg
taataaaaag agattttgag 960ctcattaaat gggtaggtgc aaattcattt
agaacatctc actatcctta tagtgaagaa 1020ataatgcagg cagctgacag
agaaggaatt gttattatag atgaagttgc tgctgtaggt 1080atgtttgatg
taggttctgt actaaatcct agtgcatcaa aaactgatta tttctcttta
1140gatgaagtac atagcaaaac taaagaagtt cataaaaaag ctgttgaaga
gcttattaaa 1200agagataaaa atcacccttc agttgttatg tggagtttat
tcaatgagcc tgatacttca 1260aaagacgaag ctgttccata ttttgaagat
atatttaatt ttgctaaaag tcaggataag 1320cagaatcttc ctaaaacttt
tgctgctatt caggcatctt ctcctggaaa atgtaagtgt 1380atgcatttat
gcgatgtaat tacattaaat agatactatg gatggtattt cttgggtggt
1440tatgaaattg atatgtcaga agaaaaattt agagaagaaa tgaatttgta
ttctaatatg 1500aataaaccag ttatgttcac agaatacgga gctgatactt
atgctggtgt tcataaactt 1560ccttccgtaa tgtggagcga agaatatcaa
tgcgaatact atgaaatgaa tttcaaagtt 1620tttgatagct atgactttat
agttggagaa caattatgga actttgctga ttttcaaact 1680actgaaggaa
tatttagggt agatggaaat aaaaaaggta tatttactag aaaccgtcag
1740cctaaagccg ttgctcatct tataagaagc agatggaata agttgccatt
agattataaa 1800tcaaaaaaa 180931823DNAArtificial SequenceOptimized
Expression of E. Coli 3ccatgggcat ggtcaatagt atgctgtatc cgcgtgaaag
ccgcacccgc cgtgttgtgg 60acatctctgg catgtgggaa ttcaaaatcg acatcaacaa
tgaaggccgt aacagtggtt 120atgcgaatgg cctgaaagat accacgttta
ttccggtccc gagctctttt aacgacctgt 180ttaccgacaa aaatatccgt
gaacatgccg gtgatgtttg gtatgaaacg agcttctacc 240tgccgctgga
atggaaagat aaagacgtga acgttcgctt tggttgcgcg acccatgaag
300ccacggtgta tattaatggc aaagaagtct gtacccacgt gggcggtttt
atgccgttca 360acgcgccggt taatgaagcc ggcatcttcg gcgagaaaaa
caaactggtg gttgtcgtga 420acaatgaact gtccaatacc acgattccgt
gcggtcacac cgaaacgaaa ccgagcggca 480aaaaatacat caaaccgtct
tttgatttct ttaactacgc aggcctgaat cgtccggtta 540aaatcaccgt
cacgaacaaa gaatacatcc atgatatcga catcctgtca gatgtgaacg
600gctcggacgg tattgtgaat tatgaagttc acaccacggg tgaaaacaaa
gtttacatca 660aaatcaacga tgaagaaggc aaagaagtcg cgagctgcga
aggcaaatct ggtaaaattg 720tgatcaaaga tgccaaactg tggaacccga
aagcggccta cctgtacaaa ttcatcgcct 780gtatcaaaaa cggtgatgaa
ctgattgacg aatattacct ggattttggc atccgcaccg 840tcaaagtgga
gggtacgaaa ttcctgatta acggcaaacc gttttatttc accggctttg
900gtaaacatga agattctgaa atcgcaggcc gtggttacaa cccgccggtt
attaaacgcg 960actttgaact gatcaaatgg gtcggtgcta atagcttccg
tacctctcac tatccgtaca 1020gcgaagaaat tatgcaggca gctgatcgcg
aaggcatcgt gattatcgac gaagttgcgg 1080ccgtcggcat gttcgatgtg
ggctctgttc tgaatccgtc agcgtcgaaa accgattatt 1140tttcactgga
cgaagttcat tcgaaaacga aagaagtcca caagaaagcg gtggaagaac
1200tgattaaacg tgataaaaac catccgagtg ttgtcatgtg gtccctgttc
aatgaaccgg 1260atacctcaaa agacgaagca gtgccgtact tcgaagatat
cttcaacttc gctaaatcgc 1320aggacaaaca aaatctgccg aaaacctttg
cagctatcca ggcgagttcc ccgggtaaat 1380gcaaatgtat gcatctgtgc
gatgtgatta cgctgaaccg ttattacggc tggtatttcc 1440tgggcggtta
cgaaatcgac atgagtgaag aaaaattccg cgaagaaatg aacctgtact
1500ccaacatgaa caaaccggtt atgttcaccg aatatggtgc agatacgtac
gctggcgttc 1560acaaactgcc gagtgtcatg tggtccgaag aataccagtg
tgaatactac gaaatgaact 1620tcaaagtgtt cgatagctac gactttattg
ttggtgaaca gctgtggaat tttgcagatt 1680tccaaaccac ggaaggcatt
tttcgcgtgg acggtaacaa gaaaggcatc ttcacccgta 1740atcgccaacc
gaaagctgtc gcacatctga ttcgctcccg ttggaacaaa ctgccgctgg
1800actacaaaag taagaagaag ctt 1823
* * * * *
References