1. expression of CYPs. Heterologous expression of human

1.
Introduction 

Cytochrome
P450 (CYP) is a haem monooxygenase that uses NADPH-cytochrome
P450 reductase (OxR) for the transfer of
electrons from NADPH to the haem moiety. It involved in major catalysis in
the oxidation of drugs, alkaloids, terpenes, pesticides, carcinogens, drug
metabolism and other xenobiotics chemicals as well as the synthesis of
cholesterol, steroids and other lipids 1. CYPs are of particular interest in
the pharmaceutical and chemical industries. CYPs have been isolated from practically
all living organisms from archaea to humans. CYP enzymes also play crucial
roles in detoxification and activation of several variety of chemicals. In
humans, CYP1, CYP2, and CYP3 families play the most important role in the
xenobiotic oxidative metabolism. One of the major human P450 enzymes is CYP3A4.
It is a 52 kDa protein and the most abundant CYP isoform in
both liver and small intestine, responsible for the oxidation of one-half the
drugs used today 2.  TBH1 

 

A
number of host cells for heterolougousheterologous expression of human CYPs are available
including mammalian cells, yeast, insect cells and bacteria. Among these
heterologous host systems, bacterial cells have advantages in terms of lower
cost to maintain, ease of use, and the high yield of protein with relatively
short period of culture time 3. Besides, they are also readily accommodating
foreign genetic material and rapid expression. Thus, the use of bacterial cells
made it possible to achieve high level expression of CYPs. Heterologous
expression of human CYP enzymes in Escherichia.
coli (E.
coli) has
been reported and been successfully served as readily available and relatively
inexpensive source of material for use in studies 3, 4. Since bacteria in
this case, E. coli,
do not possess endogenuos electron transport system to support the
full catalytic activity of CYP enzymes, co-expression of CYP and NADPH-CYP oxidoreductase (OxR) in E. coli cells has been found to be crucial to
achieve efficient catalysis and generate an active system in bacterial cells.
Moreover, CYP enzyme assays are commonly used in vitro tools to characterize kinetics of enzyme
isoforms from various sources including tissues and heterologously expressed CYPs form different host cells 5.  

 

Protein
expression can be influenced by several factors
such as the expression strain, concentration of inducer,
duration and temperature of induction
6. By increasing the
induction time, the expression rate for the target protein can be slowed down while
allowing for improved protein folding.
However, longer induction time may also induce proteolysis of the target
protein and thus reducing the yield of the expressed protein
7.  Previous study has set
the optimum temperature and induction time for CYP2D6
expressed in E. coli
at 30? C
and 24 hours, respectively 8,
while other studies have reported induction
time varying
between 24 to 72 hours for different
strains of E. coli
host cells 9According
to a similar study done by Yun et
al, the expression
levels of P450s 2A6 and 2E1 reached up to 360 and 640 nmol per liter culture at
37°C respectively within 18 h, while the levels which are consistent with those
obtained in 36 h of culturing at 28°C 9.
Hence, it is assumed as higher-temperature
over-expression systems might prove useful for the rapid generation of
substantial quantities of active human P450s 2A6 and 2E1 in E. coli.   TBH2 

 

Heterologous
expression of human CYP3A4 in E.
coli cells
was previously conducted by cloning the cDNA of CYP3A4 into pCWori+ expression
vector and by transformation into E. coli DH5? competent cells. Co-expression of OxR in E. coli was also conducted by using pACYC expression vector harbouring the cDNA of OxR due to the fact that E. coli cells do not possess endogenous electron
transport systems to support the full CYP catalytic activity 10. The presence
of antibiotic resistance gene in pCWori+ vector
and pACYC vector allow the selection and isolation of
bacterial cell populations carrying both plasmids in culture media in which
ampicillin and chloramphenicol have been incorporated. This available construct has been used to produce CYP3A4 protein for
several in vitro studies. However, the study on the optimal optimum
induction time of the expression of this construct has yet to be
carried out and thus the aims of this study. 

  

 

 

2.
Literature Review 

2.1 Cytochorme P450 (CYP) 

CYP
is a superfamily of hemoproteins, many of which can metabolize xenobiotics,
such as drugs, procarcinogens, and environmental pollutants. In humans, CYP1,
CYP2 and CYP3 families play the most important role in xenobiotic oxidative
metabolism. The majority of the human CYPs are concentrated in liver, and about
75% drug metabolism that undergoes Phase I metabolism, thus due to the
important roles of CYP in drug metabolism, activation and elimination, they
have been widely used to study drug-drug interactions 11. CYP has been
involved in the metabolism of approximately half the drugs in use today, for
example acetaminophen, codeine, cyclosporin A, diazepam and erythromycin, these drugs
can act as inducers, inhibitors or substrates. Conventional in vitro studies for human drug metabolism using
human liver microsomes, liver homogenates or hepatocyte culture are facing
several constraints and challenges such as limited tissue source, ethical issue
and concerns as well as intricate isolation and long-term storage procedures.
As a solution to these problems, heterologous expressions of the human CYPs are
made available in various host cells, including yeast, cultures mammalian
cells, insect cells and bacterial cells 12.

 

2.1.1 CYP3A4

Basically, the human CYP3A locus contains the three
CYP3A genes which are CYP3A4, CYP3A5 and CYP3A7, three pseudogenes, and a novel
CYP3A gene named CYP3A43. According to Obtsuki et al and Paine et al, hat
CYP3A4 is the most abundant and predominant hepatic form enzymes in liver composing
around 20% of hepatic CYP content and is clearly the crucial CYP enzyme present
in small intestinal enterocytes 13, 14. Thus, CYP3A4 is often considered as
most important drug-metabolizing enzyme as it has relatively high expression
level in liver and intestine and the major component of the oral
first-pass effect.  Besides,
CYP 3A7 is expressed as a major human fetal liver protein and it expressed in
placenta. CYP 3A5 is expressed in most fetal liver samples and there is general
agreement that this protein is expressed polymorphically in livers of
adolescents and adults 15. 

CYP
enzymes are commonly associated with causing many
clinically relevant drug-drug interactions 16. The ability of drugs can act as inducers,
inhibitors, or substrates for CYP3A and it might lead to altered drug disposition,
efficacy or toxicity. For
example, CYP3A4 activity can be induced or inhibited thereby changing the drug
concentrations and effectiveness 17. Microlide
antibiotics such as clarithromycin can There are drugs
that inhibit the catalytic function of CYP3A4 enzyme
and thus can interfere with the
metabolism of drugs by CYP3A4certain drugs such
as macrolide antibiotics, all of them are well-known
as inhibitors of CYP3A4 except for azithromycin. As well
asfor calcium channel blockers, it is only the non-dihydropyridine
calcium channel blockers that are the known
inhibitors of CYP3A4, but not amlodipine or nifedipine
are off the inhibitor list
18. Moreover,
CYP 3A7 is expressed as a major human fetal liver protein and it expressed in
placenta. CYP 3A5 is expressed in most fetal liver samples and there is general
agreement that this protein is expressed polymorphically in livers of
adolescents and adults. 

  

  

  

2.2 Heterologous expression of CYP 

Heterologous
expression is the expression of a foreign
protein by host cell through the cloning of complementary DNA (cDNA) or RNA
(cRNA) from another species encoding for that protein of interest.  Cells from bacteria, yeast and insects can be
used as hosts to heterologously express foreign proteins 19. Among
the many heterologous expression systems available, bacterial expression system
is the most favorable in terms of high proliferate rate, high protein yield, lo
maintenance cost, and easy handling. E.
coli is
the most extensively utilized bacterial host in the production of recombinant
human CYPs in attempt to generate ample amount of CYP proteins with relatively
short culture periods for the study on drug metabolism and toxicology. The
expression of human P450s in E.
coli has
proven to be valuable means for the characterization of these enzymes, and has
been used extensively in the production of various P450 enzymes in order to
generate large amount of the proteins 20,
21.

 

 

 

2.3 Effect of induction time on the expression
of protein  

For
successful expression of human CYPs in E. coli,
the incubation period is essential and crucial. As optimum incubation period
would allow proper expression of protein sufficiently for proper folding and heme incorporation. The induction time should be
optimized for each CYP, the optimum induction time for most of the CYP450 is 24
to 72 hours 22. According to similar study
conducted by Pritchard et
al on
the CYP2D6 content of membranes on different induction time at 30°C in E. coli cells, the induction time increaseds
gradually from 0 to 48 hours and it decreaseds
for the following hour. The optimum induction time for this study is 24 hours
that produce 1 nmol mg-1 of protein 23.   

 

2.4 Spectral activity of CYP 

According
to the Omura and Sato, the ferrous form of the hemeoprotein
reacts with carbon monoxide (CO) to form a complex which specifically has
intense absorption band at around 450 nm, thus it can readily have
detected in
dithionite band microsomes by difference spectrophotometry and it shows no
peaks other than this specific wavelengthdue to the signature
cysteine thiolate axial ligand to heme iron in the CYP 24.  The presence of this
absorption band around 450 nm
confirms the expression of
active CYP.  By using this method, it
measures the P450 CYP in
a particular biological sample indirectly and used as a parameter
to quantified the spectral content of CYP. Thus, the assays serve
as a check on the integrity of the enzyme, which is useful in routine research
activities. 

Besides, NADPH-cytochrome c reductase activity is a
useful assay to determine the activity and expression level of co-expressed OxR
25. This assay measures the reduction of cytochrome c by the reductase in the presence of
NADPH, which then form a distinct bands in the absorption spectrum.

 

  

 

 

 

 

 

 

 

3.
Research Hypotheses 

At the end of this study, the optimalum
induction time for the expression of recombinant CYP3A4 is able to be
determined. 

  

3.1
Research objectives 

  

The
objectives of this study are as follows: 

  

1.        
To determine
the optimal optimum induction
time for the expression of recombinant CYP3A4 in E. coli. 

2.        
To confirm the
expression of the recombinant CYP3A4. 

3.        
To determine
the spectral activity of the recombinant CYP3A4. 

 

3.2 Research
Questions

 

1.
What is the optimum induction time for
the expression of recombinant CYP3A4 in E. coli?

2.
What is the spectral content of CYP3A4 expressed at the optimum induction
time?

 

 

 

4.
Methodology 

4.1
Expression of recombinant CYP3A4 in E. coli 

The
glycerol stock containing the recombinant CYP3A4 will be revived by culturing
overnight in LB media supplemented with 70 µg/mL chloramphenicol and 50 µg/mL
ampicillin at 37 °C under aerobic condition. The next day, the overnight
culture will be used to inoculate 350 mL TB supplemented with the same concentration
of chloramphenicol and ampicillin as in LB media and then incubated
at 37 °C. When the culture has reached  in optical density at 600 nm wavelength, 1 mM IPTG and 0.5 mM ?-ALA will be added into the culture to
induce the expression of CYP. The CYP expression will be carried out at 30 °C
for four different induction time (16, 24, 48, 72 hours) at shaking speed of
200 rpm. 

 

4.2
Preparation of bacterial membrane 

The
growth of the 24-hour incubated culture will be arrested by chilling on ice for
10 min. After that, the culture will be harvested by centrifuging at 4 °C for
10 min at the speed of 5,000g. The pellet will be weighed before resuspended in
Tris-EDTA-sucrose (TES) buffer. Freshly prepared lysozyme
will be added into the suspension and mixed gently, then the suspension will be
diluted one-fold with chilled distilled water (dH2O), followed by incubation on
ice for 30 min with gentle shaking. After incubation, the mixture will be
centrifuged at 10,000 g for 10 min at 4 °C to collect the spheroplasts,
which will then be resuspended in spheroplasts resuspension buffer (SRB). PMSF (1 mM) and protease inhibitor (1
mL per 4 g wet cell) will be added into the SRB suspension prior to sonication.
After that, the bacterial lysate will be centrifuged at 10,000 g for 20 min at
4 °C. The supernatant will be removed carefully and subject to
ultra-centrifugation at 180,000 g for 75 min at 4 °C to collect the membranous
fractions. The membranous fraction containing the CYP and OxR proteins will be resuspended in TES-water in
a ratio of 50/50 and stored at -80 °C until further use.  

  

4.3
Determination of protein concentration 

The
protein concentration for the recombinant CYP will be determined according to
the Bradford’s method  using bovine serum
albumin at different concentrations as standard 26. According to the
Bradford’s method, a range of 5 to 100  µg protein will be prepared
with different dilutions of a protein and dilute the
unknown protein samples will be diluted to obtain 5-100 µg
protein/30 µl. Next, the standard solution or the unknown protein sample
will be added appropriately to labeled test tubes. As
for the blank sample, the standard solution will be replaced with 30 ml
dH2O. Two
blank tubes will be used, to produce the standard curve and unknown protein
samples. Few drops ofAfter that, 1 mL of
Bradford reagent will be added into each tube and mixed
well. The reaction mixtures will be Iincubated
it at room temperature for 5 min. The
absorbance will increase over time, thenbe
measured its absorbance at
595 nm using
spectrophotometer and the data will be collected to plot the standard curve8. 

 

 

   

4.4
SDS-PAGE and Western blotting analysis  

Prior
to SDS-PAGE, the protein samples will be treated with equal volume of the
sample buffer. The protein samples will
then be denatured at 95 °C for 5 min using a heat block.
After that, separation of the protein in the sample by the size will be carried
out on Mini-PROTEAN® Tetra
Cell using 10% Mini-PROTEAN® TGX™ precast gels according
to the protocols of Laemmli
27. By using micropipette, 15 mL of samples will gently havebe
loaded into
each well. Electrophoresis will be conducted out at
100V for 75 min or until the blue dye front has reached the
bottom of the polyacrylamide gel.  

           
The separated protein from the polyacrylamide gel will be
transferred onto nitrocellulose membrane through Western blotting using a
semi-dry blotting system. The nitrocellulose membrane which iswill
be pre-soaked in distilled water for 5 min and then will
be briefly soaked in transfer buffer. The blotting will be driven
at 5 V for 65 min under a constant current.  

Detection
of the blotted CYP and OxR will be carried out by using Western Max HRP
chromogenic kit. The nitrocellulose membrane will then be
first rinsed in distilled water for 5 min, followed by
wash buffer for another 5 min on a rotary shaker. The membrane will then be
put into blocking buffer for one hour at room temperature on a
rotary shaker. After that, the blocking buffer will be discarded and the
membrane will be probed with primary antibody solution (anti-human CYP3A4
monoclonal antibody)  for one hour at room temperature. The primary
antibody will then be aspirated and the blot will be washed with an ample
amount of wash buffer for 5 min with agitation. The wash buffer will be
discarded and this washing step will be repeated
twice. The membrane will be transferred into secondary antibody solution
containing HRP conjugated goat anti-lgG secondary antibody and incubated for 30 min
with gentle agitation. The secondary antibody will be discarded and the
membrane will be washed for three times with wash buffer, each for 5
min. After washing, the membrane will be put into substrate solution containing
3,3′-diaminobenzidine (DAB) substrate and incubated at
room temperature until brown colour bands are seen
on the membrane. The residual substances will be washed away by putting the
membrane in distilled water for 5 min with agitation. Finally, the membrane
will be air-dried and stored in the dark.  

  

 

 

  

  

4.5
Spectral determination of CYP content  

The CYP content of the membrane fraction will be determined
spectrophotometrically by reduced-carbon monoxide (CO) difference spectroscopy
according to the methods of Omura and Sato 124.
This method was will be adopted
in order to obtain the CYP content of the bacterial cell lysates. First, 0.150
m M phosphate buffer which contained
20% glycerol at pH 7.4 will be used
the solubilized the cell lysate. It is then Ffollowed
by diluting dilution with
0.1M phosphate buffer to obtain the final concentration of 1 g/L.
The protein sample will be distributed equally and distribute
them  aliquot into two 1 cm path
length quartz cuvettes. A few milligrams of solid dithionite will be added into
the samples and mixed with protein solution in both cuvetteswell.
By using aA UV-1650
dual-wavelength spectrophotometer will be used to
record the baseline between 400 and 500 nm.  Next, carbon
monoxide (CO) will be bubbled through the sample for 1 min and the spectrum
will be recorded.  The concentration of
CYP in the cuvette was calculated using Beer’s Law equation
as in Equation 1, where A was
the difference in light absorption at 450 nm relative to 490 nm, L was the
light path of the cuvette and the extinction coefficient e450-490
was 91 nM-1 cm-1.

 

 

A
= e
. c . L

(Equation
1)

 

The
concentration of CYP in the cuvette was calculated using Beer’s Law equation
(Equation 1).  

  

 

  

4.6
Determination of NADPH-CYP oxidoreductase activity 

A
dual-beam recording spectrophotometer will be set to time scan at a wavelength
of 550 nm. Two quartz cuvettes with I cm path length will be prepared of which
one will be served as reference cuvette and the other as sample cuvette. Each
of the cuvette will be filled with 1mL of cytochrome c solution. After that,
200 ?L of 15 mM KCN will be added into each cuvette. A
measured amount of sample protein will be added into each cuvette and mixed by
gentle inversion. The baseline absorbance versus time at 550 nm will be
recorded for 2 min. After that, a volume of 100 ?L NADPH solution will be added into the sample
cuvette and mixed gently by inversion. The increase in absorbance with time at
550 nm will be recorded over for 2 min and will be used to determine reductase
activity. The activity of
reductase was determined by Equation 2, where 0.021 is the extinction
coefficient for reduced cytochrome c and 1.2 is the total volume of reaction
mixture in cuvette. The specific content of
reductase was then calculated based on the concentration of protein in the sample.  

 

Activity
of cytochrome c reductase (nmol) = x1.2

(Equation
2)

 TBH1The
reference should be number according to their appearance in the text, thus this
reference should be renumbered as 1

 TBH2You
should talk about induction time here, not temperature