Difference between revisions of "Part:BBa K1934061"
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<h3 id="RT">1. Overproduction and purification of the p66 protein subunit</h3> | <h3 id="RT">1. Overproduction and purification of the p66 protein subunit</h3> | ||
| − | <p>The BBa_K1934061 part conceived by the 2016 INSA-Lyon team and synthesized by Genecust was cloned into pUC57 and transformed into the <i>E. coli</i> NM522 strain. One recombinant clone was grown overnight in LB at 37°C. Cells were harvested and total proteins were extracted using lysozyme and benzonase lysis protocol. After centrifugation the supernatant was collected and used for purification. The poly-His tagged p66 was purified on Ni-NTA columns from Qiagen (the protocol at the following <a href="url" https://www.qiagen.com/us/resources/resourcedetail?id=3fc8c76d-6d21-4887-9bf8-f35f78fcc2f2&lang=en> link</a>. Samples at different steps of the process were analyzed on a SDS-PAGE gel 12% and protein revealed by staining with Coomassie Blue. P66 represent 22% of the total protein produced by the recombinant NM522/pUC57- BBa_K1934061 strain (See figure below, lane | + | <p>The BBa_K1934061 part conceived by the 2016 INSA-Lyon team and synthesized by Genecust was cloned into pUC57 and transformed into the <i>E. coli</i> NM522 strain. One recombinant clone was grown overnight in LB at 37°C. Cells were harvested and total proteins were extracted using lysozyme and benzonase lysis protocol. After centrifugation the supernatant was collected and used for purification. The poly-His tagged p66 was purified on Ni-NTA columns from Qiagen (the protocol at the following <a href="url" https://www.qiagen.com/us/resources/resourcedetail?id=3fc8c76d-6d21-4887-9bf8-f35f78fcc2f2&lang=en> link</a>. Samples at different steps of the process were analyzed on a SDS-PAGE gel 12% and protein revealed by staining with Coomassie Blue. P66 represent 22% of the total protein produced by the recombinant NM522/pUC57- BBa_K1934061 strain (See figure below, lane 1). Purification on Ni-NTA of the poly-His tagged p66 allows to obtain this subunit with a purification level of 87%. </p> |
<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/3/38/INSA-Lyon_p66.jpg" width = "400"/><figcaption><b>Figure 1. Overproduction and purification of the p66 protein subunit </b> Lane 1 shows the crude cellular extract with a major band migrating at approximately 64 kDa. Such a band could is visible but attenuated in the different washing steps (lanes 2, 3, 4 and 5). Lane 6 show the purified recovered protein. The elution allows to recover 64% of the overproduced p66. P66 is pure in this fraction at 87% </figcaption></figure> | <figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/3/38/INSA-Lyon_p66.jpg" width = "400"/><figcaption><b>Figure 1. Overproduction and purification of the p66 protein subunit </b> Lane 1 shows the crude cellular extract with a major band migrating at approximately 64 kDa. Such a band could is visible but attenuated in the different washing steps (lanes 2, 3, 4 and 5). Lane 6 show the purified recovered protein. The elution allows to recover 64% of the overproduced p66. P66 is pure in this fraction at 87% </figcaption></figure> | ||
Revision as of 12:04, 19 October 2016
p66 subunit of HIV reverse transcriptase
This part contains the sequence of the p66 subunit of the HIV reverse transcriptase. It is not functional on its own and must be associated with the p51 subunit to be functional.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 238
Illegal BglII site found at 937
Illegal BglII site found at 1100
Illegal BglII site found at 1150
Illegal XhoI site found at 1800 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1739
Illegal AgeI site found at 1067
Illegal AgeI site found at 1182 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 20
Characterization
1. Overproduction and purification of the p66 protein subunit
The BBa_K1934061 part conceived by the 2016 INSA-Lyon team and synthesized by Genecust was cloned into pUC57 and transformed into the E. coli NM522 strain. One recombinant clone was grown overnight in LB at 37°C. Cells were harvested and total proteins were extracted using lysozyme and benzonase lysis protocol. After centrifugation the supernatant was collected and used for purification. The poly-His tagged p66 was purified on Ni-NTA columns from Qiagen (the protocol at the following link. Samples at different steps of the process were analyzed on a SDS-PAGE gel 12% and protein revealed by staining with Coomassie Blue. P66 represent 22% of the total protein produced by the recombinant NM522/pUC57- BBa_K1934061 strain (See figure below, lane 1). Purification on Ni-NTA of the poly-His tagged p66 allows to obtain this subunit with a purification level of 87%.
2. Assembly of a functional HIV-1 reverse transcriptase
The HIV-1 Reverse Transcriptase (RT) is a heterodimeric protein. Two subunits assemble to form the functional protein: the p51 and the p66 subunits. p66, produced and purified as described above, was mixed in PBS in equimolar ratio with p51, produced and purified from BBa K1934060. Assembly of a functional HIV-1 RT can be demonstrated by testing the enzymatic activity of the mix. Therefore, a single step RT-PCR was set, using the 16S RNA from the ribosomes as template and the ACG GCT ACC TTG TTA CGA CTT reverse and AGA GTT TGA TCC TGG CTC AG forward primers. Incubation at 40°C during 20 min enables the reverse transcription of the 16S RNA into cDNA. Then a classic PCR cycling was performed (35 cycles). The PCR mixture was then analyzed on an agarose gel (see below).