Uporaba bakteriofagnih litičnih proteinov holina in endolizina za povečano sproščanje rekombinantnih proteinov iz Lactococcus lactis NZ9000

Repar, Lara (author), Berlec, Aleš (mentor), Štravs, Petra (comentor)

Lactococcus lactis je varna mlečnokislinska bakterija, ki se uporablja kot gostiteljski organizem za izražanje terapevtsko pomembnih rekombinantnih proteinov. Pri uporabi mlečnokislinskih bakterij je ključna sposobnost sproščanja rekombinantnih proteinov iz celic. Za doseganje izločanja heterolognih proteinov se pri bakteriji Lactococcus lactis uporablja signalni peptid endogenega proteina Usp45. Prisotnost signalnega peptida je bistvena za izločanje, ki pa pogosto ni učinkovito. V okviru magistrskega dela smo vzpostavili sistem, ki temelji na izražanju aktivnih litičnih proteinov holina in endolizina endogenega laktokoknega bakteriofaga in omogoča povečano sproščanje rekombinantnih proteinov iz mlečnokislinske bakterije Lactococcus lactis NZ9000. Bakteriofagni litični protein holin v celični membrani tvori pore, ki omogočajo prehod snovi preko citoplazemske membrane. En izmed ključnih proteinov, ki ob tem preidejo celično membrano, je litični protein endolizin, ki hidrolizira peptidoglikansko celično steno. Njuno izražanje poveča permeabilnost celične membrane in razgradnjo celične stene, kar omogoča sproščanje znotrajceličnih proteinov. Za vrednotenje novorazvite metode smo pripravili genske konstrukte za sočasno izražanje litičnih proteinov z modelnim rekombinantnim proteinom celulazo, za katerega vemo, da se ne izloča učinkovito iz bakterije Lactococcus lactis. Sistem smo ovrednotili z analizo prisotnosti proteinov v celičnih lizatih in analizo sproščanja znotrajceličnih proteinov v gojišče s poliakrilamidno gelsko elektroforezo v prisotnosti denaturanta ter imunodetekcijo na membrani. Izkazalo se je, da je bilo sproščanje znotrajceličnih proteinov bistveno povečano po izražanju holina. Povečano sproščanje celulaze ob soizražanju holina smo potrdili tudi posredno z analizo sproščanja celulaze na karboksimetil celuloznih ploščah. Z analizo rasti bakterije Lactococcus lactis po izražanju litičnih proteinov smo ugotovili, da sta oba litična proteina vplivala na rast celic in podaljšala lag fazo rasti, medtem ko smo zmanjšano živost opazili le po izražanju holina. Kljub negativnemu vplivu na živost ima razviti sistem povečanega sproščanja rekombinantnih proteinov velik potencial za uporabo v biotehnologiji mlečnokislinskih bakterij.

• Language: Slovenian
• Keywords: Lactococcus lactis, sproščanje rekombinantnih proteinov, holin, endolizin, signalni peptid proteina Usp45, celulaza
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-162850
• Publication date in RUL: 28.09.2024

Secondary language

• Language: English
• Title: The use of bacteriophage lytic proteins holin and endolysin for increased release of recombinant proteins from Lactococcus lactis NZ9000

Abstract

Lactococcus lactis is safe lactic acid bacterium used as host organism for expressing therapeutically relevant recombinant proteins. When using lactic acid bacteria, their key ability is the release of recombinant proteins from cells. In Lactococcus lactis, the secretory peptide of endogenous protein Usp45 is used for enabling secretion of heterologous proteins. While the presence of signal peptide is crucial for achieving secretion, the process is not guaranteed to be effective. In the scope of this master’s thesis we established a system based on the expression of active lytic proteins, holin and endolysin, of native lactococcal bacteriophage for increased release of recombinant proteins from Lactococcus lactis NZ9000. The bacteriophage lytic protein holin forms pores in the cell membrane, enabling intracellular molecules to pass through the cytoplasmic membrane. One of the key proteins passing through the cell membrane is the lytic protein endolysin, which hydrolyses the peptidoglycan cell wall. Their expression increases the permeability of the cell membrane and cell wall degradation, causing the release of extracellular proteins. To analyse the extent of the release of the recombinant protein, we prepared genetic constructs for the co-expression of lytic proteins with the model recombinant protein cellulase. We evaluated the system by analysis of proteins in cell lysates and intracellular proteins released into the medium using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and immunodetection on the membrane. We discovered that the overall release of intracellular proteins was increased with the co-expression of holin. We also indirectly confirmed the increased release of cellulase with the co-expression of holin by analysing the release of cellulase on carboxymethyl cellulose plates. By analysing the growth and viability of Lactococcus lactis cell after the expression of lytic proteins, we found that both lytic proteins affected cell growth and delayed the lag phase, while reduced viability was observed only when holin was expressed. Despite its negative impact on viability, the developed system for enhancing the release of recombinant protein shows significant potential for application in biotechnology of lactic acid bacteria.

• Keywords: Lactococcus lactis, increased release of recombinant proteins, holin, endolysin, signal peptide Usp45, cellulase