2. Materials and Methods
Materials
All the chemicals and diaphorase used in this study were purchased from
Sigma-Aldrich Pte. Ltd., Singapore, unless otherwise stated. The QIAprep
Spin Miniprep kit for plasmid extraction was purchased from Qiagen Pte.
Ltd., Singapore. The GeneJET gel extraction kit for gel extraction was
purchased from Thermo Fisher Scientific, Singapore.
Plasmid and strain
construction
The MsfGFP gene sequence was kindly provided by Dr. Ma Lixin from Hubei
University, Wuhan, People’s Republic of China. Synthetic ADHs genes and
all the used oligonucleotides were ordered from Integrated DNA
Technologies. Plasmid construction was carried out based on the
Guanine–Thymine standard (Ma et al., 2019). The fusion proteins consist
of an N-terminal MsfGFP or eGFP which is separated by a linker with an
TEV Protease recognition site sequence (ENLYFQG) from an ADH. The DNA
sequence of all the constructed plasmids was verified by Sanger
sequencing. A full list of
plasmids constructed in this study
is provided in Table S1. E coli DH5α (C2987H, New England
Biolabs) was used as cloning host and E. coli BL21 (C2527H, New
England Biolabs) was used for protein expression. Plasmid transformation
was carried out by using the heat-shock method provided in the
manufacturer’s instruction.
Protein expression and
purification
A single colony of an E. coli BL21 strain harboring one of the
expression plasmids in Table S1 was inoculated into 3 ml of Luria
Bertani broth (LB) with 50 µg/ml spectinomycin and grown overnight at 37
°C/250 rpm. The overnight-grown culture (using 500 µl) was scaled up
with 300-fold dilution in a 500-ml shake flask and grown to a cell
density of 0.6 (optical density [OD600]) at 37 °C/250 rpm. Protein
expression was induced by adding 0.1 mM of isopropyl
β-D-1-thiogalactopyranoside (IPTG) and cells were cultured for 22 hours
at 30 °C/250 rpm.
For isolation of the intracellularly expressed (R )-2-octanol
dehydrogenase (PfODH), cells were harvested by centrifugation at 4,000 g
for 10 mins. Cell pellets were then resuspended in equilibrium buffer
(20 mM sodium phosphate, 300 mM sodium chloride, and 10 mM imidazole; pH
7.4), and mechanically lysed by a cell disrupter (OS,
Constant Systems Ltd.). The resulting cell lysate was centrifuged at
10,000 g for 20 mins to obtain the supernatant that contains soluble
proteins. For isolation of the extracellularly expressed MsfGFP fusion
proteins, the induced cell culture was centrifuged at 4,000 g for 20
mins to obtain the supernatant that accommodates secretory proteins.
Target proteins from the above two types of supernatant were both
purified by HisPur™ Ni-NTA Resin (Thermo Fisher Scientific) according to
the manufacturer’s instruction. Desalting of the protein elution was
carried out by using Sephadex G-25 PD-10 columns (GE Healthcare)
according to the manufacturer’s instruction. The concentration of
purified proteins was determined by the Micro BCA™ Protein Assay kit
(Thermo Fisher Scientific). The absorbance of assay mixtures was
measured by the Infinite 200® PRO microplate plate
reader (Tecan Group AG). The presence of the purified target proteins
was verified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using
Mini-PROTEAN TGX Precast Protein Gels (4561083, Bio-Rad). Gel image was
taken by the Gel Doc EZ Gel Documentation System (Bio-Rad).
Fluorescence observation and SDS-PAGE analysis of
supernatant
samples
The protein expression of the MsfGFP/eGFP-ADH fusion proteins was
induced as described above. The grown cell cultures were then
centrifuged at 4,000 g for 20 mins to obtain the supernatants. To
observe the fluorescence of the fusion proteins, the supernatant samples
were excited by blue light using a Safe Imager™ 2.0 Blue Light
Transilluminator (Thermo) and the image was taken through an amber
filter unit. Afterwards, the supernatant samples were concentrated
5-fold by Microcon® centrifugal filter devices (Merck Millipore) for
SDS-PAGE analysis.
The coupled enzyme assay with purified ADH/secreted fusion
proteins
The coupled enzyme assay reagent was prepared as a mixture consisting of
4 U/ml diaphorase, 1 mM resazurin, 1 mM substrate (for ADH), 1 mM
NAD+ and 0.1 M Tris buffer (pH 8.0). To examine the
catalytic activity of PfODH toward various substrates, the purified
PfODH was first diluted with 0.1 M Tris buffer to yield a final
concentration of 20 μg/ml. Subsequently, 50 µl of each diluted PfODH
sample was then pipetted into the wells of a 96-well back polystyrene
microtiter plate (Costar 3603, Corning). Assay reactions were initiated
by adding 50 µl of the assay mixture to each sample well, immediately
followed by a continuous measurement of the fluorescence of resorufin
(Ex/Em=535/588 nm) with the “Kinetic Measurement” mode in i-control
software for 10 mins by the Infinite 200® PRO
microplate plate reader (Tecan Group AG).
The same procedure was used for determining the catalytic activity of
secreted fusion proteins. Cultures of the MsfGFP-fused ADHs were
centrifuged at 4,000 g for 20 mins to obtain the supernatant that
accommodates secretory proteins. 50 µl of the supernatant sample was
used for the assay. Similarly, for high throughput screening experiment,
induced cell cultures in 96 deep well plates (Greiner Bio-One GmbH) were
centrifuged at 4,000 g for 20 mins. 50 µl of the supernatant sample from
each mutant was transferred to a well in a 96-well microtiter plate, and
the fluorescence of MsfGFP-fused mutants (Ex/Em=488/525 nm) was
determined first by plate reader. Subsequently, 50 µl of the assay
mixture was added to the well, immediately followed by a continuous
measurement of the fluorescence of resorufin (Ex/Em=535/588 nm) by plate
reader.
Kinetic analysis
Kinetic analysis of the wild-type PfODH and the selected PfODH variant
was performed by determining the production of NADH (λ = 340 nm) through
absorbance measurement. Assays were carried out in 200 μl of PBS buffer
(100 mM, pH 7.5) containing 6 mM NAD+, at room
temperature, using 13.3 μM of the wild-type PfODH or 0.055 μM of the
selected PfODH variant at substrate concentrations ranging from 0.125 mM
to 5.0 mM. The extinction coefficient used for NADH under the assay
conditions was 6220 M-1cm-1. All
assays were performed in triplicate. The enzyme kinetic parameters
km, and kcat were calculated from the
enzyme progress curves via the Lineweaver-Burk plot.