Manuscript Submitted to Bioprocess and Biosystems Engineering
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Surface Immobilization of Protein via biosilification catalyzed by
silicatein fused to glutathione S-transferase (GST)
Mi-Ran Ki, Ki Baek Yeo, and Seung Pil Pack*
Department of Biotechnology and Bioinformatics, Korea University, Jochiwon, Sejong 339700, Korea
* Corresponding author:
TEL: +82-41-860-1419, FAX: +82-41-864-2665
Email: spack@korea.ac.kr
Materials and methods
1.1 Strains, plasmids, biochemicals and chemicals
E. coli BL21 (DE3) (Agilent Technologies, Santa Clara, CA) was used as the host for the
expression system. The pET42b+ expression vector was obtained from Novagen (Madison,
WI). GFP expressing vector was kindly provided by Prof. Sun Gu Lee (Busan National
University, Korea). HRP was purchased from Worthington (Lakewood, NJ). 1-Step Slow
TMP (3, 3′, 5, 5′-tetramethylbenzidine) and GSH-coated plate were purchased from Thermo
Fisher scientific (Rockford, IL). Bis (p-aminophenoxy) dimethylsilane was purchased from
Gelest, Inc. (Morrisville, PA). Tetraethyl orthosilicate (TEOS) was purchased from SigmaAldrich (St. Louis, MO). All other reagents were of analytical grade.
1.2 Construction of expression vector
The cDNA sequence of silicatein-α of Suberites domuncular (GenBank accession number
AJ272013.1) was optimized for expression in E. coli and synthesized by Cosmo Genetech
(Seoul, Korea). The cDNA fragment for mature silicatein-α was amplified by polymerase
chain
reaction
(PCR)
with
the
forward
1
[5-
ggaattcCATATGGATTATCCGGAAGCGGTGG-3, (underlined: NdeI site)] and reverse [5ccgCTCGAGCAGGGTCGGATAGCTCGC-3,
(underlined:
XhoI
site)]
primers.
The
NdeI/XhoI digested DNA fragment cloned into likewise-digested pET42b+ vector to create
pET-SIL. The cDNA fragment for GST-fused silicatein-α was amplified by PCR with the
forward 2 [5-aaaACTAGTGATTATCCGGAAGCGGTGGATTGGCGCA-3, (underlined:
SpeI site)] and reverse [5-ccgCTCGAGCAGGGTCGGATAGCTCGC -3, (underlined: XhoI
site)] primers. The SpeI/XhoI digested DNA fragment cloned into likewise-digested pET42b+
vector to create pET-GST-SIL. The insert DNA was confirmed by gene-sequencing analysis
(Cosmo Genetech., Seoul, Korea).
1.3 Expression and purification of the fusion protein.
E. coli BL21 (DE3) harboring pET-SIL or pET-GST-SIL was grown in Terrific broth (TB)
supplemented with kanamycin (25 μg ml-1) at 37°C to an absorbance (A600) of 0.4 - 0.6,
followed by the addition of isopropyl-β-D-thiogalactoside (IPTG; 0.1 mM) and then
incubated overnight at 20°C. Soluble his-tagged silicatein or GST-silicatein (GST-SIL)
protein was purified using a HisPurTM Cobalt resin (Thermo Fisher Scientific, Rockford, IL)
according to the manufacturer’s instructions. Bound proteins were eluted with elution buffer
containing 50 mM NaH2PO4, 300 mM NaCl and 0.1 M imidazole, pH 7.6. The concentration
of imidazole has been sufficiently reduced not to interfere with the further processes using
Amicon Ultra-0.5 device according to manufacturer’s direction. Proteins were analyzed by
sodium
dodecyl
sulfate-polyacrylamide
gel
electrophoresis
(SDS-PAGE).
Protein
concentration was determined using a Bradford assay reagent (Thermo Fisher Scientific). For
immunoblotting, the resolved proteins on gel after SDS-PAGE were transferred to a PVDF membrane
by a semi-dry transfer (HorizBLOT 2M; Atto Co., Osaka, Japan). The recombinant proteins with Histag on membrane were probed with a mouse monoclonal His-tag antibody (Milipore co.) and IR-dye
800CW conjugated goat anti-mouse IgG as secondary antibody. The detection of antibody reactivity
was accomplished by Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE).
1.4 GFP purification
E. coli XL1-Blue strain harboring GFP expression vector was grown in LB medium
supplemented with ampicillin (50 μg ml-1) for 3 h at 37°C after 1 mM IPTG induction.
Soluble his-tagged GFP protein was purified in the same manner as described above.
1.5 Immobilization of GST-SIL on GSH-coated plate
After washing each GSH-coated well with phosphate buffered saline (PBS) with 0.05%
Tween-20 (PBST), 100 μl of sample containing GST-SIL was applied to each well followed
by incubation for 1 h at room temperature or overnight at 4°C. The plate was rinsed three
times with PBST. The bound GST-SIL on plate was identified by probing the protein with
anti-His tag monoclonal antibody (Millipore) and the probe was visualized by secondary
detection with IR-dye 800 CW conjugated goat anti-mouse IgG using Odyssey infrared
imaging system (LI-COR Biosciences).
1.6 Esterase activity
The esterase activity of GST-SIL was determined spectrophotometrically (at 352 nm) from
the rate of hydrolysis of Bis (p-aminophenoxy) dimethylsilane (BPDS) into p-aminophenol
(p-AP). The reaction was done at 20°C in 20 mM MOPS buffer (pH 7.6) containing 0.2 mg
ml-1 of BPDS and 2 μg of silica forming protein using a modification (5-fold reduced scale)
of the method described in the literature [1]. In control assay, silicatein or BSA or no protein
was added to reaction mixture.
1.7 Silica quantification
Silica deposition was measured by modified molybdenum blue method described by Raggi et
al[2]. First, the silified materials were dissolved in 1 M NaOH at 95°C for 30 min, followed
by adding 1M HCl for neutralization. This mixture was added to an equivalent volume of a 2
mg ml-1 ammonium haptamolybdate solution in 0.1 N sulfuric acid and incubated for 5 min.
After incubation, two equivalent volume of reducing buffer (an 1:1 mixture of 50mg ml-1
oxalic acid and 100 mM ascorbic acid in 0.3% SDS) was added. The absorbance of this
mixture was measured at 700 nm using a microplate reader (Infinite M200 PRO; TECAN,
Austria). Sodium metasilicate was used for standard silica solution.
References
[1] S.E. Wolf, U. Schlossmacher, A. Pietuch, B. Mathiasch, H.C. Schroder, W.E. Muller, W.
Tremel, Formation of silicones mediated by the sponge enzyme silicatein-alpha,
Dalton Trans 39 (2010) 9245-9249.
[2] M.A. Raggi, C. Sabbioni, R. Mandrioli, Q. Zini, G. Varani, Spectrophotometric
determination of silicate traces in hemodialysis solutions, Journal of
Pharmaceutical and Biomedical Analysis 20 (1999) 335-342.
Figure S1. Expression vector and comparison of expression levels between silicatein and
GST-SIL by immunoblot. Expression vector, pET-SIL (A) and pET-GST-SIL (B). The
codon-optimized gene of silicatein was cloned in pET42b+ at NdeI and XhoI in multi-cloning
site I for pET-SIL or cloned in pET42b+ at SpeI and XhoI sites for pET-GST-SIL,
respectively. Vector was drawn by Savy version 0.1 (http://bioinformatics.org/savvy/).
Immunoblot analysis of affinity purification eluents of silicatein (C) and GST-SIL (D)
expressed in BL21(DE3) grown at 20°C after adding 0.5 mM IPTG.
Figure S2. Efficiency of silica deposition via GST-SIL. Comparison of the efficiency of GFP
immobilization depending on the doses of silicatein or GST-SIL bound on GSH-coated
surface.
Figure S3. Effect of proteins immobilized on silica deposition rate by silicatein. The silica
deposition by 20 μg of silicatein was started by adding 100 μl of 1 M TEOS to 200 μl of 25
mM Tris-HCl (pH 6.8) buffer containing 20 μg of GFP or HRP. The reaction was done for
overnight at 20°C under vigorous shaking. The amount of silica was measured by the
molybdenum blue method.