Določanje izražanja in aktivne domene encima pernizin v bakterijah vrste Escherichia coli

magistrsko delo

Kevin Hartman (Author), Matej Butala (Reviewer), Nataša Poklar Ulrih (Mentor), Nataša Poklar Ulrih (Thesis defence commission member), Matej Butala (Thesis defence commission member), Mojca Narat (Thesis defence commission member)

Abstract

Pernizin je zunajcelična serinska proteinaza, ki jo sintetizira hipertermofilna arheja Aeropyrum pernix. Za razvoj komercialnih aplikacij in nadaljnji opis pernizina je ključen razvoj učinkovitega rekombinantnega ekspresijskega sistema. Kodonsko optimizirano zaporedje za pernizin smo vstavili v vektorje pMCSG7 in pMD204, s tem smo pripravili ekspresijske vektorje za prekomerno izražanje gena za pernizin v Escherichia coli. Optimalna čistost pernizina je bila dosežena z usmerjeno akumulacijo encima v periplazmi. Po izolaciji iz periplazemske frakcije in čiščenju encima označenega s histidinsko značko z Ni-NTA afinitetno kromatografijo je bil dosežen donos ~4 mg pernizina na L produkcijske kulture. Za aktivacijo encimske aktivnosti pernizina je potrebna avtokatalitična odcepitev proregije. Glede na predvidena cepitvena mesta post-translacijskega zorenja smo sintetizirali neprocesiran pernizin (Prn1), encim brez signalne sekvence (Prn26) ter procesiran encim brez proregije (Prn92 ; Prn94). S cimografijo smo potrdili uspešno zvitje vseh različic rekombinantnega pernizina v proteolitično aktivno obliko. To nakazuje, da proregija pri pernizinu ni nujna za uspešno zvitje, kot velja za nekatere subtilaze. S CD-spektropolarimetrijo in fluorescenčno emisijsko spektrometrijo smo potrdili visoko termostabilnost rekombinantnega pernizina ter pokazali visoko encimsko aktivnost pri temperaturi 100 °C z azokazeinskimi testi. Ugotovili smo, da med postopki produkcije in biokemijske karakterizacije v vzorcih pernizina poteka nezaželena avtoproteoliza. Vezava kalcijevih ionov (Ca2+) povzroči konformacijske spremembe v strukturi pernizina, verjetno preko tvorbe Ca2+-vezavne zanke. Vezava kalcijevih ionov zviša konformacijsko stabilnost in proteolitično aktivnost, kar posledično zviša tudi stopnjo avtokatalitičnega zorenja in avtoproteolize v raztopinah pernizina.

Keywords

pernizin;hipertermofilni encimi;Aeropyrum pernix;rekombinantno izražanje;Escherichia coli;avtokatalitično zorenje;avtoproteoliza;

Data

Language:	Slovenian
Year of publishing:	2018
Typology:	2.09 - Master's Thesis
Organization:	UL BF - Biotechnical Faculty
Publisher:	[K. Hartman]
UDC:	604.4:577.15:602.44:543.544.17(043.2)
COBISS:	8993657
Views:	995
Downloads:	290
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Determination of expression and active domain of pernisine enzyme in bacteria Escherichia coli
Secondary abstract:	Pernisine is an extracellular serine protease from the hyperthermophilic archaea Aeropyrum pernix. Development of an efficient recombinant expression system is crucial for development of industrial applications and further characterisation of this enzyme. Several expression vectors for overexpression in E. coli were constructed based on pMCSG7 and pMD204 vectors and codon-optimised pernisine gene. Initial problems with pernisine purification were resolved with translocation of enzyme accumulation into periplasm. Enzyme yield of ~4 mg L-1 production culture was achieved with isolation from the periplasmic fraction and purification of His-tagged enzyme with Ni-NTA column affinity chromatography. Pernisine is matured into an activate form with autocatalytic cleavage of proregion from the N-terminus. Based on the predicted cleavage sites of post-translational maturation unprocessed pernisine (Prn1), enzyme without signal sequence (Prn26) and maturated enzyme without the proregion (Prn92; Prn94) were synthesised. Zymography confirmed successful folding of all recombinant pernisines into an active proteolytic conformation. CD-spectrometry and intrinsic fluorescence emission spectrometry were used to demonstrate high thermal stability of recombinant pernisine and high proteolytic activity at 100 °C was shoved with azocasein assays. We have demonstrated that auto-proteolysis of pernisine samples can be problematic during enzyme production and biochemical characterisation. Binding of calcium ions (Ca2+) results in conformational changes in structure, probably via formation of Ca2+-binding loop. This increases conformational stability and proteolytic activity, thus consequently also the rate of autocatalytic maturation and auto-proteolysis in pernisine solutions.
Secondary keywords:	nanoparticles;pernisine;hyperthermophilic enzymes;autoproteolysis;
Type (COBISS):	Master's thesis/paper
Study programme:	0
Thesis comment:	Univ. v Ljubljani, Biotehniška fak., Študij biotehnologije
Pages:	XII, 64 f.
ID:	10942091