Priprava proteina CRISP iz modrasovega strupa v bakterijskem ekspresijskem sistemu

Marinko, Aljoša (author), Križaj, Igor (mentor), Gunčar, Gregor (co-mentor)

S cisteinom bogati sekretorni proteini (CRISP) so razširjeni po celotnem živalskem kraljestvu, kjer vršijo različne funkcije. So monomerni proteini z molekulsko maso od 20 do 30 kDa. Večina CRISP iz kačjih strupov je inhibitorjev ionskih kanalčkov, natančneje kalcijevih kanalčkov odvisnih od napetosti, kalijevih kanalčkov aktiviranih s kalcijem in ionskih kanalčkov aktiviranih s cikličnimi nukleotidi. Biološka funkcija CRISP iz strupa modrasa (Vipera ammodytes ammodytes, Vaa) še ni znana. Za boljše razumevanje njihove aktivnosti smo poskušali pripraviti rekombinantni VaaCRISP-1 (rVaaCRISP-1). Nukleotidno zaporedje VaaCRISP-1 je bilo vstavljeno v vektor pMAL‑c5x, ki je bil pozneje vnešen v sev bakterije Escherichia coli (E. coli) ER2523. Fuzijski protein je imel na svojem N-koncu protein, ki veže maltozo (MBP), sledil je del s cepitvenim mestom za aktivirano obliko faktorja X (FXa), nato pa rVaaCRISP‑1. MBP je omogočil lažjo izolacijo produkta iz reakcijske zmesi z afinitetno kromatografijo na imobilizirani amilozi. Izražanje fuzijskega proteina v manjšem, do četrtlitrskem obsegu je bilo uspešno, v večjem, dvolitrskem, pa ne. Medtem, ko je v prvem primeru analiza frakcije, ki se je zadržala na amilozni koloni, pokazala prisotnost fuzijskega proteina, je bil v drugem prisoten zgolj MBP. Po cepitvi fuzijskega proteina s FXa smo rVaaCRISP-1 očistili na koloni C18 sistema RP-HPLC. Glede na pridobljene strukturne karakteristike, maso in hidrofobnost, o čemer priča RP-HPLC profil, ki je enak profilu naravnega proteina, lahko s precejšnjo gotovostjo trdimo, da sta si rekombinantni in naravni VaaCRISP-1 po lastnostih enaka. Ocenjen donos izražanja v bakterijski kulturi je ~0,1 mg rVaaCRISP‑1 na liter tekočega gojišča, medtem ko imajo lahko optimizirani postopki proizvodnje rekombinantnih proteinov v citoplazmi E. coli pričakovan donos tudi nad 100 mg tarčnega proteina na liter tekočega gojišča. Razvili smo torej postopek za pripravo pravilno zvitega rVaaCRISP-1, a njegov donos bo potrebno še izboljšati.

• Language: Slovenian
• Keywords: kačji strup, Vipera ammodytes ammodytes, CRISP, rekombinantni VaaCRISP-1, E. coli
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-138065
• COBISS.SI-ID: 117051651
• Publication date in RUL: 08.07.2022

Secondary language

• Language: English
• Title: Preparation of the nose-horned viper venom protein CRISP in bacterial expression system

Abstract

Cysteine-rich secretory proteins (CRISPs) are found throughout the animal kingdom and perform different functions. They are monomeric proteins with a molar mass of 20 – 30 kDa. Most CRISPs in snake venoms are inhibitors of ion channels, including voltage‑gated K+ channels, Ca2+-dependent K+ channels, voltage-gated Ca2+ channels and ion channels regulated by cyclic nucleotides. The biological role of CRISPs in the venom of nose-horned viper (Vipera ammodytes ammodytes, Vaa) is not yet clear. To better understand their function, we have attempted to produce recombinant VaaCRISP‑1 (rVaaCRISP-1). The cDNA sequence of rVaaCRISP-1 was inserted into the vector pMAL-c5x, which was later transformed into Escherichia coli (E. coli) ER2523. The fusion protein consisted of N-terminal maltose-binding protein (MBP) to facilitate the isolation process of rVaaCRISP-1 on an amylose resin, a linker containing the Factor Xa (FXa) cleavage site and of C-terminal rVaaCRISP-1. Expression of the fusion protein on a lower scale (up to 250 ml) was successful, whereas the expression on a large (2 litre) scale failed. Namely, only analysis of the bound fraction after affinity chromatography on an amylose column of expression in the first case showed purified fusion protein. In the latter case, the analysis of the bound fraction after affinity chromatography showed only large amounts of MBP-tag and no fusion protein. After cleavage by FXa, rVaaCRISP-1 was purified by the RP-HPLC on a C18 column. Structural characteristics of rVaaCRISP-1, mass and hydrophobicity, shown by the RP‑HPLC profile, are very similar when compared to the structural characteristics of VaaCRISP-1 isolated from the crude venom. The estimated yield of our procedure was ~0,1 mg of rVaaCRISP‑1 per litre of the growth medium, while optimal procedures may give even more than 100 mg of recombinant protein per litre of growth medium. Overall, we have developed a procedure to prepare rVaaCRISP-1 in its native form, but improvements are needed to increase the yield.

• Keywords: snake venoms, Vipera ammodytes ammodytes, CRISP, recombinant VaaCRISP-1, E. coli