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.