Difference between revisions of "Part:BBa K4437003"
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__NOTOC__ | __NOTOC__ | ||
| − | <partinfo> | + | <partinfo>BBa_K4437004 short</partinfo> |
| − | NisQ with | + | This part contains the coding sequence for the antimicrobial region of NisQ tagged with a 6X-Histag, GST tag, and NusA tag. |
| − | + | __TOC__ | |
===Usage and Biology=== | ===Usage and Biology=== | ||
| + | <p>Derived from <I>Lactococcus lactis</I>, nisin is a food-safe, antimicrobial peptide (AMP) that targets a wide range of Gram-positive bacteria by binding to lipid II on the pathogens membrane, creating a pore, and causing cell death [1]. Literature suggests that nisin Q (NisQ) demonstrates greater antimicrobial and antioxidant activity against pathogens compared to other variants of nisin, such as NisA (BBa_K1365000) [2]. Nisin’s optimal pH stability is between 2 and 7 but can maintain its antibacterial activity up to a pH of 12, and can also retain its antimicrobial activity at temperatures of 120<sup>o</sup>C [2]. Unlike other AMPs, nisin is non-toxic to Gram-negative bacteria, meaning that successful recombinant expression in <I>E. coli</I> can be achieved without an inhibitory protein.</p> | ||
| + | |||
| + | ===Sequence and Features=== | ||
| + | A novel fusion protein with 6XHis-tagged Glutathione S-transferases (GST), NusA, and a 6XHis-tag fused to the N-terminus of NisQ. GST was incorporated into BBa_K4437002 due to its presence within the Xpress expression vector (BBa_K3945014), which we used to successfully produce NisQ protein. GST is a solubility tag which is useful for expressing difficult proteins. NusA is a solubility factor to help increase production and solubility of a desired peptide in E. coli. Double enterokinase cut sites are included for removal of the 6XHis-tag to isolate NisQ alone. A TEV protease site is included for removal of NusA, revealing the 6XHis-tag for subsequent NisQ purification. Using GST and NusA increases the expected band size from 7kDa (NisQ alone) to 91kDa, which is easier to visualize on SDS-PAGE gels. | ||
| + | |||
| + | ===Characterization=== | ||
| + | We used this part to successfully clone and express NisQ protein. | ||
| + | |||
| + | [[Image:DiagnosticGel.png|400px|thumb|center]] | ||
| + | <p>Figure 1. Diagnostic gel for the Xpress expression vector(BBa_K3945014) containing NisQ (within a composite part, BBa_K4437003) miniprepped-samples (samples digested at 37<sup>o</sup>C for 60 minutes with no heat inactivation because of BamHI, therefore we expected the samples to not be fully digested). Lanes 4, 5, 6, and 7 show our expected band sizes. Lane 8 contains a negative control.</p> | ||
| − | < | + | [[Image:SDS1.png|400px|thumb|center]] |
| − | + | <p>Figure 2. SDS-PAGE analysis of GST-NusA-NisQ (BBa_K4437003) samples from BL21 (DE3) E.coli strain autoinduced, using Anti-Histag antibodies and the complementary capture antibody. The process was visualised using 10% SDS-PAGE in 100V for 20 minutes and 180V for 40 minutes. Large bands in lanes 2, 4, 5, 6, and 9 at 91kDa correspond to our expected protein size.</p> | |
| − | + | ||
| + | [[Image:SDS2.png|400px|thumb|center]] | ||
| + | Figure 4. Purified SDS-PAGE of GST-NusA-NisQ (BBa_K4437003) samples samples from BL21 (DE3) E.coli strain autoinduced, using Anti-Histag antibodies and the complementary capture antibody. The process was visualised using 10% SDS-PAGE in 100V for 20 minutes and 180V for 40 minutes. Bands in lanes 3, 4, and 5 at 91kDa correspond to our expected protein size. Samples labelled "W-1" indicate wash 1, samples labelled "E-1" indicate elution 1. | ||
| − | + | ===References=== | |
| − | === | + | <ol> |
| − | < | + | <li>Zhou H, Fang J, Tian Y, Lu XY. Mechanisms of nisin resistance in Gram-positive bacteria. Annals of microbiology. 2014 Jun;64(2):413-20.</li> |
| − | < | + | <li>Mai HT, Van Hau N, Nghia NH, Thao DT. Expression and Purification of Nisin in Escherichia coli. Int. J. Life. Sci. Scienti. Res. eISSN. 2018 Jul;2455(1716):1716.</li> |
| + | </ol> | ||
Revision as of 05:35, 12 October 2022
Antimicrobial region of nisin Q
This part contains the coding sequence for the antimicrobial region of NisQ tagged with a 6X-Histag, GST tag, and NusA tag.
Usage and Biology
Derived from Lactococcus lactis, nisin is a food-safe, antimicrobial peptide (AMP) that targets a wide range of Gram-positive bacteria by binding to lipid II on the pathogens membrane, creating a pore, and causing cell death [1]. Literature suggests that nisin Q (NisQ) demonstrates greater antimicrobial and antioxidant activity against pathogens compared to other variants of nisin, such as NisA (BBa_K1365000) [2]. Nisin’s optimal pH stability is between 2 and 7 but can maintain its antibacterial activity up to a pH of 12, and can also retain its antimicrobial activity at temperatures of 120oC [2]. Unlike other AMPs, nisin is non-toxic to Gram-negative bacteria, meaning that successful recombinant expression in E. coli can be achieved without an inhibitory protein.
Sequence and Features
A novel fusion protein with 6XHis-tagged Glutathione S-transferases (GST), NusA, and a 6XHis-tag fused to the N-terminus of NisQ. GST was incorporated into BBa_K4437002 due to its presence within the Xpress expression vector (BBa_K3945014), which we used to successfully produce NisQ protein. GST is a solubility tag which is useful for expressing difficult proteins. NusA is a solubility factor to help increase production and solubility of a desired peptide in E. coli. Double enterokinase cut sites are included for removal of the 6XHis-tag to isolate NisQ alone. A TEV protease site is included for removal of NusA, revealing the 6XHis-tag for subsequent NisQ purification. Using GST and NusA increases the expected band size from 7kDa (NisQ alone) to 91kDa, which is easier to visualize on SDS-PAGE gels.
Characterization
We used this part to successfully clone and express NisQ protein.
Figure 1. Diagnostic gel for the Xpress expression vector(BBa_K3945014) containing NisQ (within a composite part, BBa_K4437003) miniprepped-samples (samples digested at 37oC for 60 minutes with no heat inactivation because of BamHI, therefore we expected the samples to not be fully digested). Lanes 4, 5, 6, and 7 show our expected band sizes. Lane 8 contains a negative control.
Figure 2. SDS-PAGE analysis of GST-NusA-NisQ (BBa_K4437003) samples from BL21 (DE3) E.coli strain autoinduced, using Anti-Histag antibodies and the complementary capture antibody. The process was visualised using 10% SDS-PAGE in 100V for 20 minutes and 180V for 40 minutes. Large bands in lanes 2, 4, 5, 6, and 9 at 91kDa correspond to our expected protein size.
Figure 4. Purified SDS-PAGE of GST-NusA-NisQ (BBa_K4437003) samples samples from BL21 (DE3) E.coli strain autoinduced, using Anti-Histag antibodies and the complementary capture antibody. The process was visualised using 10% SDS-PAGE in 100V for 20 minutes and 180V for 40 minutes. Bands in lanes 3, 4, and 5 at 91kDa correspond to our expected protein size. Samples labelled "W-1" indicate wash 1, samples labelled "E-1" indicate elution 1.
References
- Zhou H, Fang J, Tian Y, Lu XY. Mechanisms of nisin resistance in Gram-positive bacteria. Annals of microbiology. 2014 Jun;64(2):413-20.
- Mai HT, Van Hau N, Nghia NH, Thao DT. Expression and Purification of Nisin in Escherichia coli. Int. J. Life. Sci. Scienti. Res. eISSN. 2018 Jul;2455(1716):1716.