Investigation of Different Parameters Important for the Secretion of Active Indolicidin in P. pastoris

Hansen, Christian Skjødt

4. term

Nanotechnology, Master

2012

2012-06-18

77 pages

The antimicrobial peptides indolicidin and its single-tryptophan derivative indolicidin-4 can only be expressed in \textit{E. coli} by the use of fusion proteins to disable toxicity towards the host organism. However purification of active recombinant peptides is difficult and the is yield generally low. In the present study, IL and IL4 were produced in the active form by secreted expression in \textit{P. pastoris}. The productions were made in shake-flasks and fed-batch fermentations. However, the presence of recombinant peptides could not be detected in the supernatant from these productions. It was suggested that the expression was blocked by intracellular interactions or by digestion from proteolytic activity. Synthetic non-amidated IL and IL4 was found to be highly susceptible to proteases found in the supernatant. A number of attempts were made to control proteolysis, such as lowering temperature and pH and utilizing protease-deficient host organisms but without success. The importance in minimising the methanol-induced cell stress has been reported. This could be done by the controlled environment of a bioreactor equipped with a methanol substrate detector. The antimicrobial activity of compounds produced natively by \textit{P. pastoris} during methanol inductions at pH 3 has been reported in the current study. The compounds very effectively inhibited the growth of \textit{E. coli}, \textit{B. subtilis} and \textit{M. luteus}. Supernatant containing antimicrobial activity was analysed in tricine SDS-PAGE and RP-HPLC but the compounds could not be identified.

Expression ; Antimicrobial ; Peptide ; Pastoris ; Secretion ; Yield ; Recombinant ; Fermentation ; Fed-batch ; batch

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