Henrik Nausch

Recombinant Production of Human Interleukin 6 in Escherichia coli

In this study, we compared basic expression approaches for the efficient expression of bioactive recombinant human interleukin-6 (IL6), as an example for a difficult-to-express protein. We tested these approaches in a laboratory scale in order to pioneer the commercial production of this protein in Escherichia coli (E. coli). Among the various strategies, which were tested under Research and Development (R&D) conditions, aggregation-prone IL6 was solubilized most effectively by co-expressing cytoplasmic chaperones. Expression of a Glutathion-S-Transferase (GST) fusion protein was not efficient to increase IL6 solubility. Alteration of the cultivation temperature significantly increased the solubility in both cases, whereas reduced concentrations of IPTG to induce expression of the T7lac-promotor only had a positive effect on chaperone-assisted expression. The biological activity was comparable to that of commercial IL6. Targeting the expressed protein to an oxidizing environment was not effective in the generation of soluble IL6. Taken together, the presence of chaperones and a lowered cultivation temperature seem effective to isolate large quantities of soluble IL6. This approach led to in vivo soluble, functional protein fractions and reduces purification and refolding requirements caused by downstream purification procedures. The final yield of soluble recombinant protein averaged approximately 2.6 mg IL6/liter of cell culture. These findings might be beneficial for the development of the large-scale production of IL6 under the conditions of current good manufacturing practice (cGMP).

High-Level Transient Expression of ER-Targeted Human Interleukin 6 in Nicotiana benthamiana

Tobacco plants can be used to express recombinant proteins that cannot be produced in a soluble and active form using traditional platforms such as Escherichia coli. We therefore expressed the human glycoprotein interleukin 6 (IL6) in two commercial tobacco cultivars (Nicotiana tabacum cv. Virginia and cv. Geudertheimer) as well as the model host N. benthamiana to compare different transformation strategies (stable vs. transient expression) and subcellular targeting (apoplast, endoplasmic reticulum (ER) and vacuole). In T0 transgenic plants, the highest expression levels were achieved by ER targeting but the overall yields of IL6 were still low in the leaves (0.005% TSP in the ER, 0.0008% in the vacuole and 0.0005% in the apoplast). The apoplast variant accumulated to similar levels in leaves and seeds, whereas the ER-targeted variant was 1.2-fold more abundant in seeds and the vacuolar variant was 6-fold more abundant in seeds. The yields improved in subsequent generations, with the best-performing T2 plants producing the ER-targeted IL6 at 0.14% TSP in both leaves and seeds. Transient expression of ER-targeted IL6 in leaves using the MagnICON system resulted in yields of up to 7% TSP in N. benthamiana, but only 1% in N. tabacum cv. Virginia and 0.5% in cv. Geudertheimer. Although the commercial tobacco cultivars produced up to threefold more biomass than N. benthamiana, this was not enough to compensate for the lower overall yields. The recombinant IL6 produced by transient and stable expression in plants was biologically active and presented as two alternative bands matching the corresponding native protein.

Expression and subcellular targeting of human complement factor C5a in Nicotiana species.

We evaluated transgenic tobacco plants as an alternative to Escherichia coli for the production of recombinant human complement factor 5a (C5a). C5a has not been expressed in plants before and is highly unstable in vivo in its native form, so it was necessary to establish the most suitable subcellular targeting strategy. We used the strong and constitutive CaMV 35S promoter to drive transgene expression and compared three different subcellular compartments. The yields of C5a in the T0 transgenic plants were low in terms of the proportion of total soluble protein (TSP) when targeted to the apoplast (0.0002% TSP) or endoplasmic reticulum (0.0003% TSP) but was one order of magnitude higher when targeted to the vacuole (0.001% TSP). The yields could be increased by conventional breeding (up to 0.014% TSP in the T2 generation). C5a accumulated to the same level in seeds and leaves when targeted to the apoplast but was up to 1.7-fold more abundant in the seeds when targeted to the ER or vacuole, although this difference was less striking in the better-performing lines. When yields were calculated as an amount per gram fresh weight of transgenic plant tissue, the vacuole targeting strategy was clearly more efficient in seeds, reaching 35.8 µg C5a per gram of fresh seed weight compared to 10.62 µg C5a per gram fresh weight of leaves. Transient expression of C5aER and C5aVac in N. benthamiana, using MagnICON vectors, reached up to 0.2% and 0.7% of TSP, respectively, but was accompanied by cytotoxic effects and induced leaf senescence. Western blot of the plant extracts revealed a band matching the corresponding glycosylated native protein and the bioassay demonstrated that recombinant C5a was biologically active.

Public funded field trials with transgenic plants in Europe: a comparison between Germany and Switzerland

Field trails are indispensable for the scientific analysis of risks and potential benefits of genetically modified plants (GMP). The dramatic reduction of field trials in the European Union (EU) coincides with increasing safety demands, decreases in funding, and changes in the European directives. In parallel, opposition from non-governmental organizations (NGOs) has grown, and public acceptance has decreased. The cultivation of events approved by the EU is still allowed in principle, nevertheless, at least in Germany, there is a de facto moratorium on cultivation. In Switzerland, where development was much more hesitant compared to Germany, field trials are now possible, and a protected site has been established by the government. Public acceptance for scientific trials in Switzerland has risen, despite the continued moratorium on the cultivation based on a referendum.

Cyanophycinase CphE from P. alcaligenes produced in different compartments of N. benthamiana degrades high amounts of cyanophycin in plant extracts

One of the major constraints in pig and poultry farming is the supply of protein-rich forage, containing sufficient amounts of key amino acids such as arginine (Ufaz and Galili 2008). Since these are underrepresented in plant proteins, the usage of plants as feed is limited. The heterologous production of the cyanobacterial storage polymer cyanophycin granule polypeptide (CGP) in plastids increases the amount of arginine substantially (Huhns et al. 2008; Huhns et al. 2009; Nausch et al. 2016a). CGP degradation releases arginine-aspartate dipeptides. CGP is stable in plants because its degradation is exclusively restricted to bacterial cyanophycinases (CGPases; Law et al. 2009). Since animals are also unable to digest CGP, CGPases need to be co-delivered with CGP-containing plant feed in order to release the dipeptides in the gastrointestinal tract of animals during digestion. Therefore, an extracellular CGPase, CphE from Pseudomonas alcaligenes DIP-1, was targeted to the cytosol, ER, and apoplasm of Nicotiana benthamiana. Translocation to the chloroplast was not successful. Although CphE accumulated in high amounts in the cytosol, only moderate levels were present in the ER, while the enzyme was nearly undetectable in the apoplasm. This correlates with the higher instability of post-translationally modified CphE in crude plant extracts. In addition, the production in the ER led to an increased number and size of necroses compared with cytosolic expression and might therefore interfere with the endogenous metabolism in the ER. Due to the high and robust enzyme activity, even moderate CphE concentrations were sufficient to degrade CGP in plant extracts.

Expression of CphB- and CphE-type cyanophycinases in cyanophycin-producing tobacco and comparison of their ability to degrade cyanophycin in plant and plant extracts

Increasing the arginine (Arg) content in plants used as feed or food is of interest, since the supplementation of food with conditionally essential Arg has been shown to have nutritional benefits. An increase was achieved by the expression of the Arg-rich bacterial storage component, cyanophycin (CGP), in the chloroplast of transgenic plants. CGP is stable in plants and its degradation into β-aspartic acid (Asp)-Arg dipeptides, is solely catalyzed by bacterial cyanophycinases (CGPase). Dipeptides can be absorbed by animals even more efficiently than free amino acids (Matthews and Adibi 1976; Wenzel et al. 2001). The simultaneous production of CGP and CGPase in plants could be a source of β-Asp-Arg dipeptides if CGP degradation can be prevented in planta or if dipeptides are stable in the plants. We have shown for the first time that it is possible to co-express CGP and CGPase in the same plant without substrate degradation in planta by transient expression of the cyanobacterial CGPase CPHB (either in the plastid or cytosol), and the non-cyanobacterial CGPase CPHE (cytosol) in CGP-producing Nicotiana tabacum plants. We compared their ability to degrade CGP in planta and in crude plant extracts. No CGP degradation appeared prior to cell homogenization independent of the CGPase produced. In crude plant extracts, only cytosolic CPHE led to a fast degradation of CGP. CPHE also showed higher stability and in vitro activity compared to both CPHB variants. This work is the next step to increase Arg in forage plants using a stable, Arg-rich storage protein.

Paternal inheritance of plastid-encoded transgenes in Petunia hybrida in the greenhouse and under field conditions

Highlights • First proof of PMGF of a plastid-encoded transgene in the field.• Transgene integration influenced pollen quality differently in greenhouse and field.• PMGF frequency described for Petunia in the greenhouse could be verified.

An endoplasmic reticulum (ER)-directed fusion protein comprising a bacterial subtilisin domain and the human cytokine interleukin 6 is efficiently cleaved in planta

A major limitation of plant bioreactors is the lack of suitable and cost-effective purification methods for the extraction of pharmaceutical-grade proteins. In contrast to that, there are numerous established purification systems for heterologous proteins, expressed in Escherichia coli , which are used for the commercial production of therapeutic proteins. Therefore, we wanted to adapt the BioRad Profinity eXact TM one-step protein purification system (originally designed for microbial expression platforms) to purify recombinant proteins in crude plant extracts. This system based on the prodomain of microbial subtilase as fusion partner and a column-bound subtilisin protease. The engineered protease captures and cleaves the fusion protein, retaining the tag and releasing the native protein into the eluate. The subtilase tag was fused to human interleukin 6 (IL6) and transiently expressed in Nicotiana benthamiana leaves using the MagnICON system. The fusion protein was expressed at lower levels than native IL6, suggesting it is expressed less efficiently and/or has a lower stability. However, free IL6 was also detected in the extract and was unaffected by the addition of protease inhibitors during extraction, suggesting that the fusion protein is cleaved in planta by endogenous proteases. Purification of the recombinant protein using the Profinity eXact TM system reduced the yield still further. The inefficient production of tagged IL6, coupled with the extensive losses during purification, indicate that the Profinity eXact TM system is not suitable for the extraction of IL6 from crude plant extracts. Keywords : Tobacco, transient expression, endoplasmic reticulum, Profinity protein purification, partial cleavage African Journal of Biotechnology Vol. 12(3), pp. 311-319