Difference between revisions of "Part:BBa K5241002"

Latest revision as of 05:59, 29 September 2024

pkrhdd

Description: Encodes 4-hydroxyphenylpyruvate dioxygenase.

Source:Pseudomonas koreensis

Result:

1.Based on Pseudomonas_koreensis_D26 [gene=hppD][protein=4-hydroxyphenylpyruvate dioxygenase], a gene with a PelB signal peptide and 6His tag is synthesized, aiming for the tyrosinase to be localized to the bacterial periplasmic space，After constructing the plasmid and transformation, there was a small amount of melanin expressed.

2.Encodes a fusion protein consisting of the Pkrhdd enzyme from Pseudomonas koreensis, an N-terminal T7 tag for solubility, and a C-terminal 6xHis tag for purification. The fusion protein is under the control of the pBAD promoter, which allows for arabinose-inducible expression, and confers ampicillin resistance (AmpR). We used arabinose induction, and there was no visible melanin, indicating that our plasmid construction was unsuccessful.

3."PICZα" refers to a vector containing the yeast alpha-factor signal peptide sequence, which aids in the proper secretion of proteins. It was developed by Invitrogen (now part of Thermo Fisher Scientific). We used the vector pPICZαA-PHDD-6xH, which expresses the PHDD protein with a 6xHis tag in Pichia pastoris, and although colonies formed, no melanin was produced after induction with methanol, indicating that our plasmid construction failed.

Reference documentation

[1] Gätjen, D., Tomszak, F., Dettmann, J. C., Droste, M., Nölle, V., & Wieczorek, M. (2022). Design of a novel switchable antibody display system in Pichia pastoris. Applied Microbiology and Biotechnology, 106(18), 6209-6224. https://doi.org/10.1007/s00253-022-12108-5

Sequence and Features