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Revision as of 18:23, 30 September 2024
SCSdC-mCh[1-10] 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]. SCSdC-mCh[1-10], the first component of multi-polymerized MSP, is a fusion protein composed of SpyCatcher, mCherry [1-10], NW15, and SdyCatcher, 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. Through experimental validation, we added the IPTG solution at a concentration of 0.8mM when the OD value of the bacterial suspension reached 0.6-0.8, resulting in substantial expression of soluble proteins.
Figure 1.SDS-PAGE analysis of SCSdC-mCh[1-10] protein with IPTG concentration gradient induction. An IPTG concentration of 0.8mM was used for 16 hours induction at 16°C.
Purification
After confirming the successful expression of SCSdC-mCh[1-10], 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.
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.
Further Purification by SEC
Due to various possible reasons, the target protein obtained through nickel affinity chromatography has a relatively high concentration but still contains a significant amount of impurity proteins, which is detrimental to subsequent characterization experiments. Therefore, we chose to further purify the target protein to a higher degree of purity through SEC (Size Exclusion Chromatography). According to Figure 3, we obtained a total of six elution peaks. Through SDS-PAGE analysis of the samples corresponding to each peak, we confirmed that the purified target protein was obtained at the second and third peaks.
Figure 3.SEC absorption peak chromatogram of the purification results of SCSdC-mCh[1-10].
Figure 4.SDS-PAGE Analysis of SCSdC-mCh[1-10] After Further Purification by SEC. The third and fourth lanes show the presence of the target protein (corresponding to elution volumes of 56.3 and 64.55, respectively).
Structure and biological activity analysis
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 1720
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1239
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1035
Illegal AgeI site found at 1828 - 1000COMPATIBLE WITH RFC[1000]