Abstract
Exploiting secretory pathways for production of heterologous proteins is highly advantageous with respect to efficient downstream processing. In eukaryotic systems the vast majority of heterologous proteins for biotechnological application is exported via the canonical endoplasmic reticulum–Golgi pathway. In the endomembrane system target proteins are often glycosylated and may thus be modified with foreign glycan patterns. This can be destructive for their activity or cause immune reactions against therapeutic proteins. Hence, using unconventional secretion for protein expression is an attractive alternative. In the fungal model Ustilago maydis, chitinase Cts1 is secreted via an unconventional pathway connected to cell separation which can be used to co-export heterologous proteins. Here, we apply this mechanism for the production of nanobodies. First, we achieved expression and unconventional secretion of a functional nanobody directed against green fluorescent protein (Gfp). Second, we found that Cts1 binds to chitin and that this feature can be applied to generate a Gfp-trap. Thus, we demonstrated the dual use of Cts1 serving both as export vehicle and as purification tag. Finally, we established and optimized the production of a nanobody against botulinum toxin A and hence describe the first pharmaceutically relevant target exported by Cts1-mediated unconventional secretion.
Highlights
Heterologous proteins are preferentially produced in systems in which they are exported into the culture broth, as this simplifies downstream processing and minimizes the production costs substantially [1]
With the production of several different heterologous proteins like Gus, single-chain variable fragments or nanobodies [24,25], we have laid a solid foundation for follow-up studies concentrating on other relevant targets that fulfill the criteria for unconventional secretion and are hard to produce in established systems
The wells were blocked with 4% (w/v) skimmed milk in PBS for at least 4 h at room temperature and after 3× PBS-T washing, αBoNTANB-Cts1 containing cell extracts, supernatants or purified αBoNTANB-Cts1 from cell extracts or supernatants as well as the corresponding negative controls were applied to the wells either in defined volumes or protein concentrations
Summary
Heterologous proteins are preferentially produced in systems in which they are exported into the culture broth, as this simplifies downstream processing and minimizes the production costs substantially [1]. The fact that ER/Golgi-mediated co- and post-translational modifications are avoided by unconventionally secreted proteins offers new possibilities for applications in biotechnology and medicine [20,21] On this basis, we have established a novel system for protein production in the corn smut fungus Ustilago maydis. While Gus is inactivated by N-glycosylation during passage of the conventional secretion pathway of eukaryotes, it can be exported in an active state as a Cts1-fusion protein [24,28]. This confirmed that Cts1-mediated unconventional secretion avoids N-glycosylation. BAonNtiTgAen-c-boianteddinEgLaISctAivpitlyatwesa(sMtheteanbiaoslsoagyicesd, Iunsci.n, gMcaodmismone,rWciaIl, UBoSNAT).AB-icnodaitnegd aEcLtiIvSiAty pcloautelds (bMeectoabnifoirlmogeidcs,bIontch.,iMn awdhisoolne,cWelIl,eUxStrAa)c.tsBiannddinpgraoctteiivniteyncroicuhldedbefrcoomnficrumlteudrebostuhpienrnwahtaonletsceblyl eIxMtrAacCts(aFnigduprero6teCi,nDe)n. rTichhiseddefrmomoncsutrltauteres stuhpaterunnactoanntvsebnytiIoMnaAlCse(cFriegtuioren6cCa,nDb).eTehxispdloeimteodntsotrgaetensetrhaatet upnhcaornmvaecnotlioogniaclaslleycrreetlieovnancatnanbteibeoxdpylofioterdmtaotsg.enerate pharmacologically relevant antibody formats
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