Modifications of the 5' region of the CASPONTM tag's mRNA further enhance soluble recombinant protein production in Escherichia coli.

BACKGROUND: Escherichia coli is one of the most commonly used host organisms for the production of biopharmaceuticals, as it allows for cost-efficient and fast recombinant protein expression. However, challenging proteins are often produced with low titres or as inclusion bodies, and the manufacturing process needs to be developed individually for each protein. Recently, we developed the CASPONTM technology, a generic fusion tag-based platform process for high-titer soluble expression including a standardized downstream processing and highly specific enzymatic cleavage of the fusion tag. To assess potential strategies for further improvement of the N-terminally fused CASPONTM tag, we modified the 5'UTR and 5' region of the tag-coding mRNA to optimize the ribosome-mRNA interactions. RESULTS: In the present work, we found that by modifying the 5'UTR sequence of a pET30acer plasmid-based system, expression of the fusion protein CASPONTM-tumour necrosis factor α was altered in laboratory-scale carbon-limited fed-batch cultivations, but no significant increase in expression titre was achieved. Translation efficiency was highest for a construct carrying an expression enhancer element and additionally possessing a very favourable interaction energy between ribosome and mRNA (∆Gtotal). However, a construct with comparatively low transcriptional efficiency, which lacked the expression enhancer sequence and carried the most favourable ∆Gtotal tested, led to the highest recombinant protein formation alongside the reference pET30a construct. Furthermore, we found, that by introducing synonymous mutations within the nucleotide sequence of the T7AC element of the CASPONTM tag, utilizing a combination of rare and non-rare codons, the free folding energy of the nucleotides at the 5' end (-4 to + 37) of the transcript encoding the CASPONTM tag increased by 6 kcal/mol. Surprisingly, this new T7ACrare variant led to improved recombinant protein titres by 1.3-fold up to 5.3-fold, shown with three industry-relevant proteins in lab-scale carbon limited fed-batch fermentations under industrially relevant conditions. CONCLUSIONS: This study reveals some of the complex interdependencies between the ribosome and mRNA that govern recombinant protein expression. By modifying the 5'UTR to obtain an optimized interaction energy between the mRNA and the ribosome (ΔGtotal), transcript levels were changed, highlighting the different translation efficiencies of individual transcripts. It was shown that the highest recombinant titre was not obtained by the construct with the most efficient translation but by a construct with a generally high transcript amount coupled with a favourable ΔGtotal. Furthermore, an unexpectedly high potential to enhance expression by introducing silent mutations including multiple rare codons into the 5'end of the CAPONTM tag's mRNA was identified. Although the titres of the fusion proteins were dramatically increased, no formation of inclusion bodies or negative impact on cell growth was observed. We hypothesize that the drastic increase in titre is most likely caused by better ribosomal binding site accessibility. Our study, which demonstrates the influence of changes in ribosome-mRNA interactions on protein expression under industrially relevant production conditions, opens the door to the applicability of the new T7ACrare tag in biopharmaceutical industry using the CASPONTM platform process.

Ultrasound criteria for risk stratification of thyroid nodules in the previously iodine deficient area of Austria - a single centre, retrospective analysis.

BACKGROUND: We aimed to study the validity of six published ultrasound criteria for risk stratification of thyroid nodules in the former severely iodine deficient population of Austria. METHODS: Retrospective, single centre, observer blinded study design. All patients with a history of thyroidectomy due to nodules seen in the centre between 2004 and 2014 with preoperative in-house sonography and documented postoperative histology were analyzed (n = 195). A board of five experienced thyroidologists evaluated the images of 45 papillary carcinomas, 8 follicular carcinomas, and 142 benign nodules regarding the following criteria: mild hypoechogenicity, marked hypoechogenicity, microlobulated or irregular margins, microcalcifications, taller than wide shape, missing thin halo. RESULTS: All criteria but mild hypoechogenicity were significantly more frequent in thyroid cancer than in benign nodules. The number of positive criteria was significantly higher in cancer (2.79 ± 1.35) than in benign nodules (1.73 ± 1.18; p < 0.001). Thus, with a cut-off of two or more positive criteria, a sensitivity of 85% and a specificity of 45% were reached to predict malignancy in this sample of thyroid nodules. As expected, the findings were even more pronounced in papillary cancer only (2.98 ± 1.32 vs. 1.73 ± 1.18, p < 0.001). The six ultrasound criteria could not identify follicular cancer. CONCLUSION: Our findings support the recently published EU-TIRADS score. Apart from mild hypoechogenicity, the analyzed ultrasound criteria can be applied for risk stratification of thyroid nodules in the previously severely iodine deficient population of Austria.

High-resolution analysis of alterations in medullary thyroid carcinoma genomes.

Hereditary and sporadic medullary thyroid carcinoma (MTC) are closely associated with RET proto-oncogene mutations. However, the role of additional changes in the tumor genomes remains unclear. Our objective was the identification of chromosomal regions involved in MTC tumorigenesis and to assess their significance by using MTC-derived cell lines. We used array-CGH (comparative genomic hybridization) to map chromosomal imbalances in 52 primary tumors and ten metastases. Eleven tumors (11/52, 21%) were hereditary and 41 (41/52, 79%) were sporadic. Among the latter, 15 tumors (15/41, 37%) harbored RET mutations. Furthermore, we characterized five MTC cell lines in detail and evaluated the tumorigenicity by severe combined immunodeficiency (SCID)-mouse experiments. Most MTCs had only few copy number changes, and losses of chromosomes 1p, 4q, 19p and 22q were observed most frequently. The number of chromosomal aberrations increased in metastases. Twenty-three percent (12/52) of the primary tumors did not even show any chromosomal gains and losses. We injected three cell lines (two of these were without chromosomal changes and pathogenic RET mutations) into immune deficient SCID mice, and in each case, we observed rapid tumor growth at the injection sites. Our data suggest that MTCs--in contrast to most other tumor entities--do not acquire a multitude of genomic imbalances. SCID mouse experiments performed with chromosomally normal cell lines and without RET mutations suggest that presently unknown submicroscopic genomic changes are sufficient in MTC tumorigenesis.

Improved downstream process for the production of plasmid DNA for gene therapy.

Gene therapy and genetic vaccines promise to revolutionize the treatment of inherited and acquired diseases. Since viral vectors are generally associated with numerous disadvantages when applied to humans, the administration of naked DNA, or DNA packed into lipo- or polyplexes emerge as viable alternatives. To satisfy the increasing demand for pharmaceutical grade plasmids we developed a novel economic downstream process which overcomes the bottlenecks of common lab-scale techniques and meets all regulatory requirements. After cell lysis by an in-house developed gentle, automated continuous system the sequence of hydrophobic interaction, anion exchange and size exclusion chromatography guarantees the separation of impurities as well as undesired plasmid isoforms. After the consecutive chromatography steps, adjustment of concentration and final filtration are carried out. The final process was proven to be generally applicable and can be used from early clinical phases to market-supply. It is scaleable and free of animal-derived substances, detergents (except lysis) and organic solvents. The process delivers high-purity plasmid DNA of homogeneities up to 98% supercoiled form at a high yield in any desired final buffer.

Prevalence of autoimmune thyroiditis and non-immune thyroid disease in multiple sclerosis.

Since multiple sclerosis (MS) and autoimmune thyroiditis (AIT) are presumed to be of autoimmune origin the correlation of these two diseases is of special interest. The aim of this study was to determine whether there are differences in the prevalence of thyroid disease with special emphasis on AIT compared with MS and normal subjects and whether the presence of thyroid disease correlates with disability, disease course, age, and disease duration. 353 consecutive patients with clinically definite MS, without interferon-beta treatment and 308 patients with low back pain or headache were extensively examined for the presence of non-immune or autoimmune thyroid disease. We found a significantly higher prevalence of AIT in male MS patients (9.4 %) than in male controls (1.9 %; p = 0.03). The prevalence of AIT in female MS patients (8.7 %) did not differ from female controls (9.2 %). Hypothyroidism, caused by AIT in almost all cases, showed a tendency to be more severe and more often present in patients with MS. There was no association between relapsing-remitting and secondary progressive disease course of MS and the prevalence of AIT. MS patients with AIT were significantly older but did not differ in disease duration and expanded disability status scale (EDSS). Further studies are warranted, to see if there is a difference in sex-hormone levels between MS patients with and without AIT and healthy controls. Longitudinal studies comparing MS patients with or without AIT could show whether there is an influence of AIT on the disease course or progression.