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

Undersøgelse af Forskellige Parametre Vigtig for Sekreringen af Aktiv Indolicidin i P. pastoris

Hansen, Christian Skjødt

4. term

Nanotechnology, Master

2012

2012-06-15

77

Antimikrobielle peptider som indolicidin kan dræbe bakterier, men de er også skadelige for almindelige produktionsværter som Escherichia coli. For at undgå denne toksicitet fremstilles de typisk som fusionsproteiner (midlertidigt bundet til et bærerprotein), hvilket gør oprensning vanskelig og giver lave udbytter. I dette studie blev det undersøgt, om aktivt indolicidin (IL) og dets enkelt‑tryptofan‑variant indolicidin‑4 (IL4) kunne produceres ved sekretion fra gæren Pichia pastoris. Kulturer blev dyrket i rystekolber og i fed‑batch‑fermenteringer, men der kunne ikke påvises rekombinant IL eller IL4 i supernatanten (væsken uden for cellerne). Fraværet af målpeptider kan skyldes intracellulære interaktioner, der blokerer sekretion, eller nedbrydning af proteaser (enzymer der kløver proteiner). Syntetiske, ikke‑amiderede IL og IL4 var stærkt følsomme over for proteaser i P. pastoris‑supernatant. Forsøg på at begrænse denne proteolyse—ved at sænke temperatur og pH samt bruge protease‑defekte værtsstammer—lykkedes ikke. Da metanol bruges til at udløse produktion i P. pastoris og kan stresse cellerne, er det vigtigt at minimere metanol‑induceret stress; bedre kontrol kan opnås i en bioreaktor udstyret med en metanol‑substratdetektor. Interessant nok producerede P. pastoris under metanol‑induktion ved pH 3 naturlige forbindelser med stærk antimikrobiel aktivitet, som hæmmede væksten af Escherichia coli, Bacillus subtilis og Micrococcus luteus. Analyse af aktive supernatantprøver med tricine SDS‑PAGE (proteingel) og omvendt fase HPLC kunne ikke identificere de ansvarlige forbindelser. Samlet set viser resultaterne, at sekretion af indolicidin‑familiepeptider fra gær er udfordrende, og at strammere proceskontrol og beskyttelse mod proteaser kan være nødvendig.

Antimicrobial peptides such as indolicidin can kill bacteria, but they are also harmful to common production hosts like Escherichia coli. To avoid this toxicity, they are usually made as fusion proteins (temporarily attached to a carrier), which makes purification difficult and yields low. This study sought to produce active indolicidin (IL) and its single‑tryptophan variant indolicidin‑4 (IL4) by secreting them from the yeast Pichia pastoris. Cultures were run in shake flasks and fed‑batch fermentations, but no recombinant IL or IL4 was detected in the culture supernatant (the liquid outside the cells). The lack of detectable peptide may be due to intracellular interactions that block secretion or to degradation by proteases (protein‑cutting enzymes). Synthetic, non‑amidated IL and IL4 were highly susceptible to proteases present in P. pastoris supernatants. Attempts to limit this proteolysis—by lowering temperature and pH and by using protease‑deficient host strains—did not succeed. Because methanol is used to trigger production in P. pastoris and can stress the cells, minimizing methanol‑induced stress is important; better control may be achievable in a bioreactor equipped with a methanol substrate detector. Interestingly, during methanol induction at pH 3, P. pastoris produced native compounds with strong antimicrobial activity that inhibited Escherichia coli, Bacillus subtilis, and Micrococcus luteus. Analyses of active supernatants by tricine SDS‑PAGE (protein gel) and reverse‑phase HPLC did not identify the responsible compounds. Overall, these results underscore the challenge of secreting indolicidin‑family peptides from yeast and point to the need for tighter process control and protection from proteases.

[This abstract was generated with the help of AI]

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

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