The production of recombinant T4 endonuclease V in Pichia pastoris fermentation
Date Issued
2016
Date
2016
Author(s)
Lin, Yu-Jiun
Abstract
T4 endonuclease V (T4N5) is a DNA repair enzyme isolated from T4 bacteriophage. It can specifically recognize and remove cyclobutane pyrimidine dimer which is resulted from the UV-induced DNA damage and might cause skin cancer. In previous reports, in conjunction of recombinant T4N5 expressed by Escherichia coli and liposome, T4N5 liposome lotion effectively penetrated into skin in vivo and clinical trials also indicated its potential for UV-induced DNA damage treatment. In order to extensive application of T4N5 in skin care products such as sunscreen, it’s important to develop a method for large-scale production recombinant T4N5 with low expenses. The major recombinant T4N5 are currently produced by E. coli. However, the procedures include cell lysis and protein purification, which may increase the production cost. Moreover, it is at risk due to endotoxins. Pichia pastoris is a well-defined expression system which can be cultivated at high cell density at low cost. In this study, P. pastoris KM71H was used as a heterologous expression system, and methanol was added to induce recombinant T4N5 expression. In flask, we have successfully expressed extracellular T4N5 with correct conformation and function. The T4N5 activity in supernatant of flask culture reached up to 0.47±0.05 U/µL. When using 5 L fermenter, methanol fed at 2.6 mL//L/h induced cell lysis and release of cellular proteases, while reducing methanol feeding rate led to low yield of target protein yield. Therefore, it’s necessary to modify other induction conditions. In this study, we found that reduction of the induction temperature from 30oC to 23oC improved recombinant protein yield remarkably at the same methanol feeding rate. The amount of proteases in the culture decreased, and methanol dissimilation is improved.
Subjects
Recombinant T4 endonuclease V
Fermentation
Methanol induction
Low-temperature cultivation
SDGs
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
ntu-105-R03b22022-1.pdf
Size
23.32 KB
Format
Adobe PDF
Checksum
(MD5):52234a872bd2d8d25a01fee27f7531da