2.1. Preparation of recombinant proteins
Genes encoding superfolder green fluorescent protein (sfGFP) and LipM7,
a lipase recently discovered in our laboratory via metagenomics analysis
(Table S1, to be published elsewhere) were cloned into the plasmid
pET21a (Novagen, Wisconsin, USA) between the Nde I and Xho I
restriction enzyme sites to generate pET21a-sfGFP and pET21a-LipM7,
respectively. For construction of plasmids encoding N-terminal phasin
fusion proteins (PhaP-sfGFP and PhaP-LipM7), phasin gene phaPfrom Aeromonas hydrophila (accession number, UniprotKB-O32470)
was synthesized by GenScript (Nanjing, China) and inserted between theHind III and Xho I sites of pET21a-sfGFP or pET21a-LipM7. To
prepare recombinant proteins fused with phasin at the C-terminus
(sfGFP-PhaP and LipM7-PhaP), sfGFP and LipM7 genes were first cloned
into the pET21a vector between the Nde I and Hind III sites,
followed by insertion of the phasin sequence between the Hind III
and Xho I sites (see Table S2 for primers used for plasmid
construction).
Recombinant proteins were prepared from cultures of Escherichia
coli strain BL21 (DE3) (Novagen, Darmstadt, Germany) transformed with
corresponding plasmids. Cells were grown at 37°C in a 2 L baffled flask
containing 400 mL of Luria-Bertani medium supplemented with 50 µg/mL of
ampicillin. When the absorbance at 600 nm (OD600)
reached 0.5 to 0.6, the culture broth was supplemented with 0.5 mM
isopropyl-β-D-thiogalactopyranoside (IPTG), and incubated at 20°C for a
further 20 h. Cells were harvested by centrifugation (6,800 × g ,
20 min) and lysed by sonication in 10 mL of 50 mM sodium phosphate
buffer (pH 8.0) containing 300 mM NaCl, 10 mM imidazole, and 1 mg/mL
lysozyme. The supernatant of the centrifuged lysate (6,800 × g ,
20 min) was loaded onto a column containing 2 mL Ni-NTA agarose resin
(Qiagen, Hilden, Germany). After extensive washing with 20 mM imidazole
in 50 mM sodium phosphate buffer, resin-bound proteins were eluted with
250 mM imidazole solution (1 mM imidazole for sfGFP-PhaP and
PhaP-sfGFP). The eluate was dialyzed against 100 mL of 20 mM sodium
phosphate buffer (pH 7.4). After adjusting to a protein concentration of
0.5 mg/mL, the protein solutions were stored at 4°C prior to use.
The purity of proteins was analyzed using 12% sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). The sizes of resolved
proteins were estimated using PM2700 Protein Markers (SMOBIO, Hsinchu,
Taiwan). Protein concentration was determined by the Bradford assay
following the manufacturer’s protocol (BioRad, Hercules, CA, USA).
Fluorescence of sfGFP, sfGFP-PhaP, and PhaP-sfGFP was measured using a
VICTOR X2 plate reader (Perkin Elmer, Waltham, MA, USA) set at
wavelengths of 485 and 535 nm for excitation and emission, respectively.
The enzymatic activity of LipM7 lipase was determined by a colorimetric
method using p -nitrophenyl decanoate (pNPD) as a substrate, as
described previously (Choi et al., 2013).