Investigation of methylotrophic yeasts expression systems for the production of self-assembling proteins with biological applications

Bredell, Wilhelmina Jacoba (2018-12)

Thesis (PhD)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Functional biological proteins are gaining interest with potential application in medicine and biotechnology. The methylotrophic yeasts, especially Pichia pastoris and Hansenula polymorpha, have been successfully used for the production of heterologous proteins with high industrial and medicinal importance. The aim of this study was to explore the production of self-assembly proteins in methylotrophic yeasts. The specific proteins investigated were the Human Papilloma virus (HPV) and Rotavirus (RV) proteins that self-assemble into virus-like particles (VLPs), and the α-chain proteins that assembles into procollagen. Cervical cancer is ranked the fourth most common cancer in women worldwide. Human Papilloma virus types 16 and 18 account for 70% of all cervical cancers worldwide. Despite the availability of two commercial prophylactic vaccines, it is unaffordable for most women in developing countries. We compared the optimized expression of monomers of the unique HPV type 16 L1/L2 chimeric protein (SAF) in two yeast strains of P. pastoris, KM71 (MutS) and GS115 (Mut+), together with H. polymorpha NCYC 495, to determine the preferred host in bioreactors. SAF was uniquely created by replacing the h4 helix of the HPV type 16 capsid L1 protein with a 13-amino acid peptide from the L2 protein. Two different methanol-feeding strategies in fed-batch cultures of P. pastoris MutS were evaluated: a predetermined feed rate versus feeding based on the oxygen consumption by maintaining constant dissolved oxygen levels (DO stat). All cultures showed a significant increase in biomass concentrations when methanol was fed using the DO stat method. In P. pastoris, the SAF concentrations were higher in the MutS strains than in the Mut+ strains. Our results showed the maximum concentration SAF expressed in both yeasts reported to date with H. polymorpha the best producer of all the yeasts evaluated. Further, accurate quantification of SAF was obtained through direct comparison with known concentrations of purified HPV16 L1. Quantification is usually inferred from ELISA results using standards not always compatible to the L1 monomer. Previous research in insect cells showed that the HPV16 L1/L2 chimeric protein self-assembled in capsomeres or capsomer aggregates during expression in insect cells. With the human codon-optimized chimeric gene, occational T=7 VLPs were visible in the insect cells. Similarly, when SAF was expressed in both methylotrophic yeasts, the majority of heterologous protein was observed as capsomeres (10 nm in diameter) with the occasional T=1 VLPs (25-30 nm in diameter) assembled. This is the first report showing the formation of T=1 VLPs in H. polymorhpa. SAF proteins displaying L2 epitopes offer simultaneously high titres of L1 specific neutralizing antibodies, as well as cross-neutralizing antibodies against L2. Gastroenteritis is one of the leading causes of deaths in children under the age of five years worldwide and caused by RV. We expressed a RV VP6 protein, derived from a prevalent South African RV strain (G9P), intracellularly in Escherichia coli, P. pastoris and H. polymorpha. Despite producing the lowest biomass levels of all the expression systems in shake flasks, the highest VP6 concentration was obtained with E. coli. In the controlled environment of bioreactors, all three expression systems attained higher cell densities and increased growth-associated VP6 production, in comparison to shakeflasks. Unlike in shake flask expressions, H. polymorpha outperformed both P. pastoris and E. coli during bioreactor cultivation. In contrast to yeast expressions, bacterial expressed VP6 protein was found to be insoluble upon analysis. This is the first report of VP6 expressed in methylotrophic yeast and holds the promise for the inexpensive production of VP6 as a possible vaccine candidate, booster dose or drug delivery mechanism. Collagen is the main structural protein of various animal connective tissues and also has the natural ability to self-assemble; therefor, it poses similar expression challenges than VLPs. Treatment of burn victims can benefit from combining a collagen α-chain with an antimicrobial peptide (AMP) in wound dressings. The collagen can aid in wound healing and the AMP in fighting infectious agents present. This collagen-AMP fusion protein was extracellularly produced by H. polymorpha. Presence of a correctly-sized single band on tricine-SDS PAGE gels revealed the successful expression of the putative fusion protein. Proteomic analysis of this protein species only identified part of this fusion. This is the first evidence of the possible successful expression of a recombinant collagen-AMP fusion protein in yeast. This study suggests that H. polymorpha is the preferred host, among the host cells tested, for the production of self-assembly proteins, such as the protein-components of virus-like particles and structural fusion proteins, such as collagen-AMP.

AFRIKAANSE OPSOMMING: Funksionele biologiese proteïene met potensiële toepassing in medisyne en biotegnologie wek al hoe meer belangstelling. Metilotrofiese giste, veral Pichia pastoris en Hansenula polymorpha, word suksesvol aangewend vir die vervaardiging van heteroloë proteïene met hoë industriële en medisinale belang. Die doel van hierdie studie was om die produksie van self-monterende proteïene in metilotrofiese giste te ondersoek. Die spesifieke proteïene wat ondersoek is, was die Menslike Papilloomvirus (MPV) en Rotavirus (RV) proteïene wat self-monteer in virusagtige partikels (VAPs), asook die α-kettingproteïene in prokollageen. Servikale kanker is die vierde mees algemene kanker by vroue wêreldwyd. Menslike Papilloomvirus tipes 16 en 18, verteenwoordig 70% van alle servikale kankers wêreldwyd. Ten spyte van die beskikbaarheid van twee kommersiële voorkomende entstowwe, is dit vir die meeste vroue in ontwikkelende lande onbekostigbaar. Ons het die geoptimiseerde uitdrukking van die unieke MPV tipe 16 L1 / L2 chimeriese proteïen (SAF) monomere in twee gisstamme van P. pastoris, KM71 (MutS) en GS115 (Mut+), vergelyk tesame met H. polymorpha NCYC 495, om die voorkeurgasheer in bioreaktors te bepaal. SAF was uniek geskep deur die h4-helix van die HPV tipe 16 kapsied L1 proteïen met 'n 13-aminosuurpeptied van die L2 proteïen te vervang. Twee verskillende metanolvoedingstrategieë in P. pastoris MutS kulture is geëvalueer: 'n voorafbepaalde voertempo versus voeding gebaseer op die suurstofverbruik deur konstante opgeloste suurstofvlakke (OS) te handhaaf. Alle kulture het 'n beduidende toename in biomassa getoon toe metanol met behulp van die OS metode gevoer is. Die SAF konsentrasies was hoër in P. pastoris MutS-kulture as in Mut+-kulture. Ons resultate toon dat albei giste die maksimum konsentrasie SAF wat gerapporteer is, oortref het met H. polymorpha wat die hoogste vlakke het van al die giste wat geêvalueer was. Verder is akkurate kwantifisering van SAF verkry deur direkte vergelyking met bekende konsentrasies van suiwer HPV16 L1. Kwantifisering word gewoonlik afgelei uit ELISA resultate deur gebruik te maak van standaarde wat nie altyd met L1 monomere ooreenstem nie. In vorige studies in insekselle het hierdie HPV16 L1 / L2 chimeriese proteïen self gemonteer om kapsomere of kapsomeer-aggregate te vorm tydens uitdrukking. Met die menskodon-geoptimiseerde chimeriese geen was T = 7 VAPs soms sigbaar in insekselle. Soortgelyk, toe SAF in beide metilotrofiese giste uitgedruk is, was die meerderheid heteroloë proteïen as kapsomere (10 nm in deursnee) waargeneem met hier en daar ‘n T = 1 VAP (25-30 nm in deursnee). Dit is die eerste verslag wat die vorming van T = 1 VAPs in H. polymorhpa demonstreer. SAF proteïene wat L2-epitope vertoon, bied gelyktydig hoë titers van L1-spesifieke neutraliserende teenliggame, sowel as kruis-neutraliserende teenliggame teen L2. Gastro-enteritis is wêreldwyd een van die grootste oorsake van sterftes by kinders onder die ouderdom van vyf jaar en word deur RV veroorsaak. Ons het 'n RV VP6 proteïen, gebasseer op 'n Suid-Afrikaanse RV stam (G9P), intrasellulêr in Escherichia coli, P. pastoris en H. polymorpha uitgedruk. Ten spyte van E. coli se lae biomassavlakke in skudflesse, het hierdie uitdrukkingsisteem die hoogste VP6 konsentrasie gelewer. In vergelyking met skudflesse, het al drie uitdrukkingstelsels in die beheerde omgewing van bioreaktors tipiese hoër seldigtheid en groeiverwante VP6 produksie gelewer. Anders as in skudflesuitdrukkings, het H. polymorpha beter as beide P. pastoris en E. coli in bioreaktor kulture presteer. In teenstelling met gisuitdrukkings, was bakteriële uitgedrukte VP6 proteïene onoplosbaar. Dit is die eerste verslag van VP6 wat in metilotrofiese giste uitgedruk is en toon belofte vir bekostigbare VP6 produksie wat as moontlike entstofkandidaat kan dien, of as aanvullende vaksien of die moontlike draer van sekere teikenmedisyne. Kollageen is die hoof strukturele proteïen van verskeie diere bindweefsels en het ook die natuurlike vermoë om self te monteer; dus stel dit soortgelyke uitdagings as VAPs om uitgedruk te word. Behandeling van brandslagoffers kan baat vind by die kombinasie van 'n kollageen-α-ketting met 'n antimikrobiese peptied (AMP) in wondbedekkings. Die kollageen kan help met wondgenesing en die AMP om aansteeklike patogene te beveg. Hierdie kollageen-AMP-fusie-proteïen is ekstrasellulêr in H. polymorpha vervaardig. Teenwoordigheid van die korrekte grootte proteïenband op trisien-SDS PAGE-jels het die suksesvolle uitdrukking van die vermeende fusie-proteïen bevestig. Proteomiese analise van hierdie proteïenspesie het slegs ‘n gedeelte van die fusie proteïen geïdentifiseer. Nietemin, dit is die eerste bewys van die moontlike suksesvolle uitdrukking van 'n rekombinante kollageen-AMP-fusie-proteïen in gis. Hierdie studie dui daarop dat H. polymorpha die voorkeurgasheer is vir die produksie van self-monterende proteïene, soos die proteïen-komponente van virusagtige deeltjies, asook strukturele fusieproteïene, soos kollageen-AMP.

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