Difference between revisions of "Part:BBa K5321017"
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| − | <partinfo>BBa_K5321017 | + | ===Sequence and Features=== |
| + | <partinfo>BBa_K5321017 SequenceAndFeatures</partinfo> | ||
| + | ===Usage and Biology=== | ||
Maltose-binding protein (MBP) tag is a large (43 kDa) periplasmic and highly soluble protein of E. coli that acts as a solubility enhancer tag. It also increases protein expression levels. Considering of the large molecular weight of MBP, we were concerned that the MBP tag may interfere with the binding of SA and biotin. So we added a TEV-cutsite between the MBP and SA. | Maltose-binding protein (MBP) tag is a large (43 kDa) periplasmic and highly soluble protein of E. coli that acts as a solubility enhancer tag. It also increases protein expression levels. Considering of the large molecular weight of MBP, we were concerned that the MBP tag may interfere with the binding of SA and biotin. So we added a TEV-cutsite between the MBP and SA. | ||
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| + | '''Figure 1 | Overview of plasmid construction for streptavidin-biotin interactions based crosslinking.'''<br> | ||
| + | ===Characterization=== | ||
| + | ====Protein Purification==== | ||
| + | Next, we transferred the correct plasmid into the expression strain of *E.coli* BL21(DE3). When the culture reached an OD600 of approximately 0.55 at 37°C, IPTG was added, and the induction was carried out overnight at 20°C. Then we did Protein Solubility Verification. We found that our protein was predominantly located in the inclusion bodies, but a portion was also present in the supernatant. The we used nickel beads to purify protein from supernatant. However, there were little target protein in eluate. | ||
| − | + | So, we can only obtain our protein from the inclusion bodies. Since the majority of the protein in the inclusion bodies is our target protein, We directly denatured the washed inclusion body protein using 8M urea, followed by gradient dialysis for refolding. Then we cleaved the refolded protein with TEV overnight. | |
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| + | '''Figure 2 | SDS analysis of the protein purified from supernatant and washed inclusion bodies for SA_cPPVp, SA_nPPVp, MBP_SA_cPPVp and MBP_SA_nPPVp.''' Lane1-4, protein purified from supernatant. From lane 1 to 4, MBP_SA_cPPVp, MBP_SA_nPPVp, SA_cPPVp and SA_nPPVp, respectively. Lane5-8, washed inclusion bodies. From lane 5 to 8, MBP_SA_cPPVp, MBP_SA_nPPVp, SA_cPPVp and SA_nPPVp, respectively. | ||
| − | + | ====Ligation Reaction==== | |
| − | + | We added different amounts of cleaved MBP_SA_nPPVp to 5'biotin-29mer and MBP_SA_cPPVp to 5'biotin-40mer. After incubating at 37°C for half an hour, we performed EMSA to verify the link between protein and nucleic. The EMSA results showed that as the amount of protein increased, the nucleic acid bands darkened. However, no shifted bands were observed. | |
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| − | + | '''Figure 3 | EMSA analysis of the protein-nucleic linkage.''' Lane1-4, 5'biotin-29mer and cleaved MBP_SA_cPPVp. Only add aptamer in lane 1. From lane 2 to 5, protein amounts for lane are 0.3ug, 0.6ug, 1.2ug, 2.4ug, respectively. Lane6-10, 5'biotin-40mer and cleaved MBP_SA_nPPVp. Only add aptamer in lane 6. From lane 7 to 10, protein amounts for lane are 0.9ug, 1.8ug, 3.6ug, 7.2ug, respectively. The amounts for aptamer is 20pmol each lane. | |
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Latest revision as of 14:36, 22 September 2024
MBP_SA_cPPVp
Contents
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 405
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1705
Illegal AgeI site found at 1387 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 103
Usage and Biology
Maltose-binding protein (MBP) tag is a large (43 kDa) periplasmic and highly soluble protein of E. coli that acts as a solubility enhancer tag. It also increases protein expression levels. Considering of the large molecular weight of MBP, we were concerned that the MBP tag may interfere with the binding of SA and biotin. So we added a TEV-cutsite between the MBP and SA.
Figure 1 | Overview of plasmid construction for streptavidin-biotin interactions based crosslinking.
Characterization
Protein Purification
Next, we transferred the correct plasmid into the expression strain of *E.coli* BL21(DE3). When the culture reached an OD600 of approximately 0.55 at 37°C, IPTG was added, and the induction was carried out overnight at 20°C. Then we did Protein Solubility Verification. We found that our protein was predominantly located in the inclusion bodies, but a portion was also present in the supernatant. The we used nickel beads to purify protein from supernatant. However, there were little target protein in eluate.
So, we can only obtain our protein from the inclusion bodies. Since the majority of the protein in the inclusion bodies is our target protein, We directly denatured the washed inclusion body protein using 8M urea, followed by gradient dialysis for refolding. Then we cleaved the refolded protein with TEV overnight.
Figure 2 | SDS analysis of the protein purified from supernatant and washed inclusion bodies for SA_cPPVp, SA_nPPVp, MBP_SA_cPPVp and MBP_SA_nPPVp. Lane1-4, protein purified from supernatant. From lane 1 to 4, MBP_SA_cPPVp, MBP_SA_nPPVp, SA_cPPVp and SA_nPPVp, respectively. Lane5-8, washed inclusion bodies. From lane 5 to 8, MBP_SA_cPPVp, MBP_SA_nPPVp, SA_cPPVp and SA_nPPVp, respectively.
Ligation Reaction
We added different amounts of cleaved MBP_SA_nPPVp to 5'biotin-29mer and MBP_SA_cPPVp to 5'biotin-40mer. After incubating at 37°C for half an hour, we performed EMSA to verify the link between protein and nucleic. The EMSA results showed that as the amount of protein increased, the nucleic acid bands darkened. However, no shifted bands were observed.
Figure 3 | EMSA analysis of the protein-nucleic linkage. Lane1-4, 5'biotin-29mer and cleaved MBP_SA_cPPVp. Only add aptamer in lane 1. From lane 2 to 5, protein amounts for lane are 0.3ug, 0.6ug, 1.2ug, 2.4ug, respectively. Lane6-10, 5'biotin-40mer and cleaved MBP_SA_nPPVp. Only add aptamer in lane 6. From lane 7 to 10, protein amounts for lane are 0.9ug, 1.8ug, 3.6ug, 7.2ug, respectively. The amounts for aptamer is 20pmol each lane.