Difference between revisions of "Part:BBa K3153002"
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<h2>Protocol </h2> | <h2>Protocol </h2> | ||
| + | SDS-PAGE | ||
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| + | A 12% SDS-PAGE gel was prepared, as the expected molecular weight of SpPCS protein was 46Kda. Then 5μl marker or 20μl protein samples were loaded in each lane. Run SDS-PAGE and the electrophoresis was complete when the the dye front migrates about 2mm from the bottom of the gel. The gel was stained with Coomassie brilliant blue for 30min, then then destained in destain buffer overnight. | ||
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| + | WB | ||
| + | A 12% SDS-PAGE gel was prepared and 5μl marker or 20μl protein samples were loaded in each lane. At the end of SDS-PAGE, the gel was transferred to PVDF membrane in WB transfer buffer for 2h. Then the PVDF membrane was washed in TBST and blocked in WB blocking buffer for 1h. After blocking, the membrane was washed in TBST and incubated with Rabbit-anti-Histag antibody diluted 1:10,000 in blocking buffer at 4℃overnight. The next day the membrane was washed in TBST three times and incubated with goat-anti-rabbit IgG conjugated with HRP diluted 1:1000 in blocking buffer at RT for 1h. After washing three times in TBST, the membrane was exposed in the darkroom with film developer solution and the WB photos was obtained. | ||
Revision as of 09:15, 21 October 2019
oprF-fMT
A surface-displayed fusion protein for aresenic removal. OprF, the major outer membrane (OM) protein of Pseudomonas aeruginosa, has been used for presentation of foreign protein on the cell surface. fMT,is an arsenic-chelating metallothionein (fMT) from the arsenic-tolerant marine alga Fucus vesiculosus
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 608
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 697
- 1000COMPATIBLE WITH RFC[1000]
Background
Metallothionein (MT) plays an important role in bioremediation of heavy metals. MT is a kind of small molecular peptide rich in cysteine (Cys-) residues with low molecular weight. Its Cys- residues can adsorb many heavy metals such as lead, mercury and cadmium. However, since arsenic is a metalloid, these MTs have no specific adsorption on it.
Recently, scientists isolated and identified an MT from a species of arsenic-tolerant Marine algae (Fusus vesiculosus) that, in vitro, demonstrated a high affinity for arsenic binding capacity.
In this project, we used the bacterial surface display technology to display fMT on the bacterial surface, and thus enhance the ability of absorbing arsenic.
Inspired by HUST-China team in 2014, the fusion protein oprf-fMT was constructed and heterogeneiously expressed by the E. coli expression system pET series plasmid.
Protocol
SDS-PAGE
A 12% SDS-PAGE gel was prepared, as the expected molecular weight of SpPCS protein was 46Kda. Then 5μl marker or 20μl protein samples were loaded in each lane. Run SDS-PAGE and the electrophoresis was complete when the the dye front migrates about 2mm from the bottom of the gel. The gel was stained with Coomassie brilliant blue for 30min, then then destained in destain buffer overnight.
WB A 12% SDS-PAGE gel was prepared and 5μl marker or 20μl protein samples were loaded in each lane. At the end of SDS-PAGE, the gel was transferred to PVDF membrane in WB transfer buffer for 2h. Then the PVDF membrane was washed in TBST and blocked in WB blocking buffer for 1h. After blocking, the membrane was washed in TBST and incubated with Rabbit-anti-Histag antibody diluted 1:10,000 in blocking buffer at 4℃overnight. The next day the membrane was washed in TBST three times and incubated with goat-anti-rabbit IgG conjugated with HRP diluted 1:1000 in blocking buffer at RT for 1h. After washing three times in TBST, the membrane was exposed in the darkroom with film developer solution and the WB photos was obtained.
Result
As shown in figure, the photograph of SDS-PAGE and Western blot of orpF-fMT fusion protein was successfully expressed under IPTG induction with the molecular weight between 25KDa and 35kda, which was consistent with the expected molecular weight of 27.4kda, indicating that the fusion protein was successfully expressed.
Fig.1 IPTG-induced protein expression of recombinant plasmid pET24a-oprF-fMT in SDS-PAGE M：protein marker, 1-3 for oprF-fMT fusion protein expression, and 1: IPTG(-); 2: IPTG (1mM), 37℃,6h; 3: IPTG(1mM), 25℃,overnight; (b) IPTG-induced protein expression of recombinant plasmid pET24a-oprF-fM in Western blot by using anti-Histag antibody.