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 secretion signal peptide (mating factor alpha) was fused to the HBD2 peptide to direct the S. cerevisiae chassis to secrete the peptide out into culture media, thereby facilitating the ease of HBD2 purification. 6xHis-Tag has been fused to the C-terminus of the peptide to enable the ability to perform Ni-NTA column purification of HBD2 from the culture media.
A suitable promoter and terminator is required for this part.
Design
Due to problems associated with expressing recombinant lactoferrin in S. cerevisiae, an alternative antimicrobial peptide (AMPs) was required as a candidate biopesticide. After extensive research into literature and consulting with companies exploring the use of AMPs for agriculture use, Beta-defensins were chosen. Vaciome, a Singaporean synbio startup focusing on AMPs for livestock protection, agreed that in theory, defensins could function as robust antifungals. Jonathan Bester, CEO of Vaciome, strongly believed this project had strong potential to impact the future of crop protection and generously donated to a Human Beta Defensin 2 (HBD2) plasmid optimized for P.pastoris.
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.
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.
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.
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