Difference between revisions of "Part:BBa K5301007"

Revision as of 06:57, 1 October 2024

SnTST-mCh[11] is one of the components of multi-polymerized MSP.

Usage and Biology

In order to produce large nanodiscs more conveniently, we hope to flexibly extend the length of MSP according to demand, and thus propose the concept of multi-polymer MSP, which refers to large circular MSPs through end-to-end connections of multiple MSP fragments. We used NW15 as the basic MSP and selected three types of linkers (Spy/Sdy/Snoop) to achieve the connection of different MSP fragments through the formation of covalent bonds, and adopted rigorous design to prevent self-cyclization of each fragment of the multi-polymer MSP. Finally, the successful cyclization of large circular MSPs is characterized by the fluorescence of mCherry after the combination of mCherry [1-10] and mCherry [11]. SnTST-mCh[11], the third component of multi-polymerized MSP, is a fusion protein composed of SnoopTag, NW15, mCherry [11], and SpyTag, with flexible GS linkers used to connect each part.

Cultivation, Purification and SDS-PAGE

Induction

We chose the T7 expression system as the pathway for protein expression and induced protein expression by adding IPTG at an appropriate time. To achieve efficient expression of SnTST-mCh[11], we screened the induction conditions using IPTG. We set up a gradient of four IPTG concentrations: 0.2mM, 0.5mM, 0.8mM, and 1mM. The results showed that the optimal protein expression was achieved with a concentration of 0.8mM.

Figure 1.SDS-PAGE analysis of SnTST-mCh[11] protein with IPTG concentration gradient induction. An IPTG concentration gradient of 0.2mM, 0.5mM, 0.8mM, and 1mM was used for 16 hours induction at 16°C. The molecular weight of SnTST-mCh[11] is 33.6 kDa.

Purification

After confirming the successful expression of SnTST-mCh[11], we scaled up the culture and purified the protein. During plasmid construction, we incorporated a His-tag into the sequence, allowing for purification using nickel affinity chromatography, which specifically binds to His-tagged proteins. We eluted the protein using 100mM and 500mM imidazole, and successfully obtained target protein. To verify the successful expression of the protein, we conducted a Western Blot using anti-Histag after SDS-PAGE and confirmed that we had obtained SnTST-mCh[11] with a relatively high purity.

Figure 2.Western Blot Analysis of SCSdC-mCh[1-10] Purified by Nickel Affinity Chromatography. The presence of the target protein in the 100mM imidazole elution buffer was verified through the staining of Histag antibody and the correct molecular weight of the bands. The molecular weight of SnTST-mCh[11] is 33.6 kDa.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 757
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 276
Illegal BglII site found at 648
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 ===Purification===
-After confirming the successful expression of SnTST-mCh[11], we scaled up the culture and purified the protein. During plasmid construction, we incorporated a His-tag into the sequence, allowing for purification using nickel affinity chromatography, which specifically binds to His-tagged proteins. We eluted the protein using 300mM and 500mM imidazole, respectively, and obtained a large amount of target protein in both cases, as shown in figure 2.
+After confirming the successful expression of SnTST-mCh[11], we scaled up the culture and purified the protein. During plasmid construction, we incorporated a His-tag into the sequence, allowing for purification using nickel affinity chromatography, which specifically binds to His-tagged proteins. We eluted the protein using 100mM and 500mM imidazole, and successfully obtained target protein. To verify the successful expression of the protein, we conducted a Western Blot using anti-Histag after SDS-PAGE and confirmed that we had obtained SnTST-mCh[11] with a relatively high purity.
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 https://static.igem.wiki/teams/5301/parts/tri-1-purification.png
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-Figure 2.SDS-PAGE Analysis of SCSdC-mCh[1-10] Purified by Nickel Affinity Chromatography. Both 300mM and 500mM imidazole elution resulted in the acquisition of a significant amount of target protein. The molecular weight of SnTST-mCh[11] is 76.3 kDa.
+Figure 2.Western Blot Analysis of SCSdC-mCh[1-10] Purified by Nickel Affinity Chromatography.  The presence of the target protein in the 100mM imidazole elution buffer was verified through the staining of Histag antibody and the correct molecular weight of the bands. The molecular weight of SnTST-mCh[11] is 33.6 kDa.
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