Part:BBa_K3927018
mFA-HBD2
Coding sequence for the human immune protein, human beta defensin 2 fused to the mating factor alpha peptide for secretion in S.cerevisiae
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 85
Description
This part is a composite part comprising of part BBa_K792002 (Mating Factor Alpha) and part BBa_K3927017 (Human Beta Defensin 2).
Usage
A suitable promoter and terminator is required for successful expression of this part. HBD2 is designed to be secreted out of the S. cerevisiae chassis for ease of purification.
Recommended induction time if using a strong inducible promoter is 72 - 120 hours, depending on the culture size and incubation conditions.
Once induction phase is complete, separate culture media from cells via centrifugation. Purify culture supernatant using Ni-NTA column to isolate expressed HBD2.
Design
The HBD2 fragment has been fused with mating factor alpha to allow for the secretion of expressed peptide into the culture media for ease of purification. No cell lysis is required to extract out HBD2, however, amount of intracellular HBD2 is greater that secreted HBD2 due to secretion inefficiencies of the chassis.
Purification from the culture media can be performed using Ni-NTA spin/FPLC columns.
Characterisation
The HBD2 fragment was obtained by amplifying the HBD2 gene from the P. pastoris plasmid donated by Vaciome. Using Gibson Assembly, the HBD2 fragment was assembled into a plasmid with a Gal1 promoter upstream, with a 6xHis-tag fused to the C-terminus. The new construct was named pGmHBD2-H, and was transformed into BY4741 to test the expression, secretion, and functionality of HBD2 via galactose induction (Figure 1).
Figure 1: Schematic diagram of construct pGmHBD2-H.
pGmHBD2-H was inoculated from glycerol stock in YPD-HygB and incubated overnight. Culture media was swapped to YPGR to initiate galactose induction. After 48 hours, the culture was spun down to isolate the media supernatant and the cell pellet. The media was concentrated by 100x and purified using centrifugal Ni-NTA columns. Cell pellet was washed and lysed, after which it was centrifuged, and the lysate supernatant was retained. SDS-Page was performed on the media, media concentrate, purified media, and media concentrate, as well as unpurified and His-tag purified cell lysates.
Figure 2: Coomasie blue staining of the media and cell lysate containing induced pGmHBD2-H. 8kDa band corresponding to size of human beta defensin 2 was observed in all lanes except the wildtype negative control.
To test the functionality of the HBD2 produced, we performed a MIC assay using the agar diffusion method against E. coli to validate the HBD2 function with the MIC data from Vaciome. The total protein expression of HBD2 was subpar in comparison to the MIC data that Vaciome produced using HBD2 expressed from P. pastoris culture. A small zone of inhibition was produced only when we concentrated our media isolate by 100x, in contrast, Vaciome’s P. pastoris media isolate had enough HBD2 to produce a zone of inhibition when used neat. A possible explanation is that S. cerevisiae was not as efficient in expression and secretion of HBD2 as compared to P. pastoris.
Figure 3: MIC carried out with samples isolated from pGmHBD2-H. Top left: Flow through from His column purification, Top right: 1mg/ml Ampicillin positive control, Bottom left: Unconcentrated media, Bottom right: 100x concentrated media from His purification.
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