Difference between revisions of "Part:BBa K4623012"
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===Purification of Basic Silinker=== | ===Purification of Basic Silinker=== | ||
| + | After successfully obtaining the PS protein, it is necessary to scale up the cultivation and perform purification of the target protein. We induced large-scale expression using 0.1 mM IPTG at 16°C for 16-20 hours to produce a significant amount of the target protein. His-tag affinity chromatography was utilized for purification. As shown in the following figure, a substantial amount of the target protein was successfully eluted using 500 mM imidazole, resulting in prominent bands. | ||
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===Functional examination of PS proteins=== | ===Functional examination of PS proteins=== | ||
==Functional examination of PS proteins== | ==Functional examination of PS proteins== | ||
Revision as of 11:41, 10 October 2023
Pairing Silinker, dimerization in response to specific signals on the silica surface
Usage and Biology
The Pairing Silinker (PS) is a novel recombinant protein that efficiently binds to the surface of silica and undergoes dimerization in response to specific signals on the silica surface. The sequence in the FASTA format includes an added HIS tag, enabling purification of the PS protein using a nickel column. Upstream of the sequence, a TrxA** (part number)** fusion tag is added to aid protein folding and reduce the formation of inclusion bodies in bacterial cells. Following protein expression, cleavage by thrombin** (part number)** exposes the mSA** (part number)** site, allowing binding to a biotinylated functional protein. The SBP** (part number)** sequence can bind to the silica surface, facilitating the modification of functional proteins onto the silica surface.
After introducing the pETDuet1 plasmid into our engineered BL21(DE3) bacteria, we conducted a small-scale trial expression and found that BL21(DE3) exhibited high basal expression levels, leading to the formation of PS inclusion bodies in uninduced strains. Therefore, in subsequent experiments, we employed the BL21(DE3) pLysS strain, which carries the pLysS plasmid and expresses T7 lysozyme to suppress leaky expression caused by T7 RNA polymerase. Experimental results demonstrated a significant increase in the solubility of PS.
Purified Pairing Silinker can be detected via SDS-PAGE, with a molecular weight of approximately 44 kDa, higher than the theoretical value of 34 kDa. This discrepancy may be attributed to flexible C-terminal sequences like SBP. To enhance the purification strategy, we have also developed corresponding hardware, utilizing the binding affinity between SBP and silica to purify the protein. This greatly improves the efficiency of protein production and purification, reducing costs.
Contents
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 445
Illegal AgeI site found at 505 - 1000COMPATIBLE WITH RFC[1000]
Cultivation, Purification and SDS-PAGE
induction condition
The presence of mSA monomers indeed tends to promote protein aggregation and inclusion body formation, making purification more challenging. To achieve efficient expression of PS while reducing inclusion body formation, we conducted tests on the culture conditions of our BL21(DE3) engineered bacteria. We set up four different culture media: Media A: LB + 50 mM KH2PO4, 50 mM Na2HPO4, 25 mM (NH4)2SO4, and 2 mM MgSO4; A + 50 mM mannitol; A + 2% ethanol; A + 4% glycerol. Experimental results showed that the addition of media additives did not significantly reduce inclusion body formation or improve protein solubility expression. Furthermore, the control group indicated another issue: BL21(DE3) exhibited excessive leaky expression, as evident from the presence of prominent inclusion bodies even in uninduced bacterial cells.
Purification of Basic Silinker
After successfully obtaining the PS protein, it is necessary to scale up the cultivation and perform purification of the target protein. We induced large-scale expression using 0.1 mM IPTG at 16°C for 16-20 hours to produce a significant amount of the target protein. His-tag affinity chromatography was utilized for purification. As shown in the following figure, a substantial amount of the target protein was successfully eluted using 500 mM imidazole, resulting in prominent bands.