Optimizing expression of Nanobody® molecules in Pichia pastoris through co-expression of auxiliary proteins under methanol and methanol-free conditions.

BACKGROUND: Ablynx NV, a subsidiary of Sanofi, has a long-standing focus on the development of Nanobody® molecules as biopharmaceuticals (Nanobody® is a registered trademark of Ablynx NV). Nanobody molecules are single variable domains, and they have been met with great success part due to their favorable expression properties in several microbial systems. Nevertheless, the search for the host of the future is an ongoing and challenging process. Komagataella phaffi (Pichia pastoris) is one of the most suitable organisms to produce Nanobody molecules. In addition, genetic engineering of Pichia is easy and an effective approach to improve titers. RESULTS: Here we report that P. pastoris engineered to co-express genes encoding four auxiliary proteins (HAC1, KAR2, PDI and RPP0), leads to a marked improvement in the expression of Nanobody molecules using the AOX1 methanol induction system. Titer improvement is mainly attributed to HAC1, and its beneficial effect was also observed in a methanol-free expression system. CONCLUSION: Our findings are based on over a thousand fed-batch fermentations and offer a valuable guide to produce Nanobody molecules in P. pastoris. The presented differences in expressability between types of Nanobody molecules will be helpful for researchers to select both the type of Nanobody molecule and Pichia strain and may stimulate further the development of a more ecological methanol-free expression platform.

Pichia pastoris Mut(S) strains are prone to misincorporation of O-methyl-L-homoserine at methionine residues when methanol is used as the sole carbon source.

BACKGROUND: Over the last few decades the methylotrophic yeast Pichia pastoris has become a popular host for a wide range of products such as vaccines and therapeutic proteins. Several P. pastoris engineered strains and mutants have been developed to improve the performance of the expression system. Yield and quality of a recombinant product are important parameters to monitor during the host selection and development process but little information is published regarding quality differences of a product produced by different P. pastoris strains. RESULTS: We compared titer and quality of several Nanobodies(®) produced in wild type and Mut(S) strains. Titer in fed-batch fermentation was comparable between all strains for each Nanobody but a significant difference in quality was observed. Nanobodies expressed in Mut(S) strains contained a product variant with a Δ-16 Da mass difference that was not observed in wild type strains. This variant showed substitution of methionine residues due to misincorporation of O-methyl-L-homoserine, also called methoxine. Methoxine is likely synthesized by the enzymatic action of O-acetyl homoserine sulfhydrylase and we confirmed that Nanobodies produced in the corresponding knock-out strain contained no methoxine variants. We could show the incorporation of methoxine during biosynthesis by its addition to the culture medium. CONCLUSION: We showed that misincorporation of methoxine occurs particularly in P. pastoris Mut(S) strains. This reduction in product quality could outweigh the advantages of using Mut strains, such as lower oxygen and methanol demand, heat formation and in some cases improved expression. Methoxine incorporation in recombinant proteins is likely to occur when an excess of methanol is present during fermentation but can be avoided when the methanol feed rate protocol is carefully designed.

Stimulation of Toll-like receptor 3 and 4 induces interleukin-1beta maturation by caspase-8.

The cytokine interleukin (IL)-1beta is a key mediator of the inflammatory response and has been implicated in the pathophysiology of acute and chronic inflammation. IL-1beta is synthesized in response to many stimuli as an inactive pro-IL-1beta precursor protein that is further processed by caspase-1 into mature IL-1beta, which is the secreted biologically active form of the cytokine. Although stimulation of membrane-bound Toll-like receptors (TLRs) up-regulates pro-IL-1beta expression, activation of caspase-1 is believed to be mainly initiated by cytosolic Nod-like receptors. In this study, we show that polyinosinic:polycytidylic acid (poly[I:C]) and lipopolysaccharide stimulation of macrophages induces pro-IL-1beta processing via a Toll/IL-1R domain-containing adaptor-inducing interferon-beta-dependent signaling pathway that is initiated by TLR3 and TLR4, respectively. Ribonucleic acid interference (RNAi)-mediated knockdown of the intracellular receptors NALP3 or MDA5 did not affect poly(I:C)-induced pro-IL-1beta processing. Surprisingly, poly(I:C)- and LPS-induced pro-IL-1beta processing still occurred in caspase-1-deficient cells. In contrast, pro-IL-1beta processing was inhibited by caspase-8 peptide inhibitors, CrmA or vFLIP expression, and caspase-8 knockdown via RNAi, indicating an essential role for caspase-8. Moreover, recombinant caspase-8 was able to cleave pro-IL-1beta in vitro at exactly the same site as caspase-1. These results implicate a novel role for caspase-8 in the production of biologically active IL-1beta in response to TLR3 and TLR4 stimulation.

Adenoviral gene transfer of ABIN-1 protects mice from TNF/galactosamine-induced acute liver failure and lethality.

Tumor necrosis factor (TNF) is a proinflammatory cytokine that plays a central role in acute and chronic hepatitis B and C infection and alcoholic liver disease as well as fulminant liver failure. TNF-induced liver failure is characterized by parenchymal cell apoptosis and inflammation leading to liver cell necrosis. The transcription factor NF-kappaB is believed to mediate at least part of the proinflammatory effects of TNF, and is therefore a favorite drug target. However, NF-kappaB also suppresses TNF-mediated hepatocyte apoptosis, implicating a potential cytotoxic effect of NF-kappaB inhibitors in the liver. This dual function of NF-kappaB emphasizes the need for therapeutics that can inhibit both TNF-induced NF-kappaB activation and cell death. Here we describe that adenoviral expression of the NF-kappaB inhibitory protein ABIN-1, but not an IkappaBalpha superrepressor (IkappaBalpha(s)), completely prevents lethality in the TNF/D-(+)-galactosamine-induced model of liver failure. Protection was associated with a significant decrease in TNF-induced leukocyte infiltration as well as hepatocyte apoptosis. The differential effects of ABIN-1 and IkappaBalpha(s) suggest a role for an NF-kappaB independent function of ABIN-1. Indeed, ABIN-1 was found to prevent not only NF-kappaB activation, but also apoptosis of cultured hepatocytes in response to TNF, explaining its protective effect against TNF-induced liver failure. In conclusion, ABIN-1 has a dual NF-kappaB inhibitory and anti-apoptotic activity in the liver, which might be of considerable interest for the treatment of inflammatory liver diseases.

Targeting Rac1 by the Yersinia effector protein YopE inhibits caspase-1-mediated maturation and release of interleukin-1beta.

Yersinia bacteria can take control of the host cell by injecting so-called Yop effector proteins into the cytosol of the cells to which they adhere. Using Yersinia enterocolitica strains that are deficient for one or more Yops, we could show that YopE and, to a lesser extent, YopT interfere with the caspase-1-mediated maturation of prointerleukin-1beta in macrophages. In addition, overexpression of YopE and YopT was shown to prevent the autoproteolytic activation of caspase-1 in a way that is dependent on their inhibitory effect on Rho GTPases. Expression of constitutive-active or dominant-negative Rho GTPase mutants or treatment with Rho GTPase inhibitors confirmed the role of Rho GTPases and, in particular, Rac1 in the autoactivation of caspase-1. Rac1-induced caspase-1 activation was mediated by its effect on LIM kinase-1, which is targeting the actin cytoskeleton. Rac-1 and LIM kinase-1 dominant-negative mutants were shown to inhibit caspase-1 activation induced by overexpression of Asc, which is a caspase-1-activating adaptor protein. Moreover, Rac1 as well as YopE and YopT significantly modulated caspase-1 oligomerization. These results highlight a previously unknown function of Rho GTPases in the activation of caspase-1 and give new insight on the role of YopE in immune-escape mechanisms of Yersinia.

The yeast three-hybrid system as a tool to study caspases.

Caspases are cysteine proteases that play an essential role during apoptotic cell death and inflammation. They are synthesized as catalytically dormant proenzymes, containing an N-terminal prodomain, a large subunit (p20) containing the active site cysteine, and a small subunit (p10). The active enzymes function as tetramers, consisting of two p20/p10 subunit heterodimers. Both subunits contribute residues that are essential for substrate recognition. Activation of caspases culminates in the cleavage of a set of cellular proteins, resulting in disassembly of the cell or proinflammatory cytokine production. Inappropriate caspase activation contributes to or accounts for several diseases. The identification of caspase-interacting proteins that might act as activators, substrates, or inhibitors is therefore an attractive step in the development of novel therapeutics. However, caspase substrates and other proteins that bind specifically with the active heterodimeric p20/p10 form of caspases will escape detection in a classical two-hybrid approach with an unprocessed caspase precursor as bait. Alternatively, a number of so-called three-hybrid systems to analyze more complex macromolecular interactions have been developed. We describe the use of a three-hybrid approach adapted to the needs of caspases to detect and analyze the interaction of mature heteromeric caspases with protein substrates or inhibitors.

Caspase-11 gene expression in response to lipopolysaccharide and interferon-gamma requires nuclear factor-kappa B and signal transducer and activator of transcription (STAT) 1.

Murine caspase-11, together with caspase-1, is essential for the production of IL-1beta in response to lipopolysaccharide (LPS). In most cells, caspase-11 is only expressed upon induction with pro-inflammatory stimuli. To understand how caspase-11 expression is transcriptionally regulated, we isolated the caspase-11 gene promoter by genome walking and investigated the mechanisms regulating caspase-11 gene expression in macrophages that are treated with LPS and interferon-gamma. Transient transfections with caspase-11 promoter-luciferase reporter constructs and deletion/mutation analysis revealed an essential role for NF-kappaB binding in the up-regulation of caspase-11 in response to LPS. In the case of interferon-gamma stimulation, signal transducer and activator of transcription 1 binding to the caspase-11 promoter could be shown to be required for caspase-11 expression.

Reversion of autoimmune lymphoproliferative syndrome with an antimalarial drug: preliminary results of a clinical cohort study and molecular observations.

Autoimmune lymphoproliferative syndrome (ALPS) is a paediatric disease characterized by lymphoproliferation and autoimmunity. Most patients are known to carry heterozygous mutations of the TNFRSF6 gene leading to diminished Fas-mediated apoptosis and failure of activated lymphocytes to undergo apoptosis. A subgroup of patients without the TNFRSF6 gene mutation has similar defective apoptosis and clinical features. No effective treatment has been reported so far. Glucocorticoids, intravenous immunoglobulin and/or immunosuppressive drugs have usually led to only transient clinical improvement. Seven ALPS patients (two type Ia and five type III) were treated with the antimalarial drug Fansidar. No toxicity was observed. An objective response was seen in six of them and, in two, the treatment was stopped without reappearance of the symptoms. Moreover, a marked decrease in interleukin-10 levels was observed in two patients during the treatment. We found that the drug induced apoptosis in activated lymphocytes through activation of the mitochondrial apoptotic pathway.