Part:BBa_K4429002
Full Length Anti-MBP Recombinant IgY
Generates a full-length chimeric IgG-IgY antibody against maltose-binding protein in when cloned into a pET-21b vector. Check design page for more specific information.
Characterisation
CLONING
A 0.8% agarose gel was run with the suspected positive clones along with the undigested empty vector. The positive clones are expected to show an upward shift of about 2200 bp. As a further test for confirmation, a restriction digest with HindIII was run. HindIII is a single cutter for the positive clone, and a non-cutter for the empty vector. A 0.8% agarose gel was run with the products of the restriction digest. A positive clone would be linearized by HindIII. We were successful in cloning the gene for the IgY-IgG into our vector. We have confirmed the same by having the plasmid sequenced.
Expression check
We observed bands of varying intensities corresponding to the different concentrations of IPTG and determined that a IPTG concentration of 0.5mM is ideal for maximum yield of protein.
PURIFICATION
The culture was grown at 16℃ overnight (14-16 hours) and spun down at 4C, 5000 rpm, for 15 mins. The pellet was resuspended using Lysis buffer and were sonicated (at 60% amplitude with a 2 sec ON, 5 sec OFF cycle) until the solution turned slightly brown and translucent. The solution was then spun down (12000 rpm for 25 minutes at 4C), and the supernatant, which contained our protein, was purified using Ni-NTA beads and eluted using increasing concentrations of Imidazole. The flow-through, the different elutes, and cracked beads was run on a 12% SDS-PAGE to resolve the different protein bands along with a protein ladder that shows 10 bands with 3 reference bands.
For the anti-MBP IgY antibody, we got fragmented bands when we ran a SDS-PAGE. This was possibly be due to working with an improper protease inhibitor.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 739
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 739
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 739
- 1000COMPATIBLE WITH RFC[1000]
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