In vivo and in vitro Cleavage of Glucoamylase-TNFα Fusion Protein Secreted from Aspergillus niger

The most common expression strategy for secreting heterologous proteins from filamentous fungus Aspergillus niger is based on fusion with glucoamylase gene which contains cleavage site for kexin protease (KEX2). However, secretion of recombinant proteins in the form of a fusion-protein without a host-specific cleavage site is usually higher than secretion of the mature protein obtained after in vivo cleavage. We tried to take advantage of such a higher production by cleaving the fusion protein in vitro after fermentation, instead of in vivo during secretion. Similar level of production as after in vivo cleavage was found when human tumor necrosis factor α (TNFα) was produced as a fusion protein with glucoamylase having the enterokinase cleavage site. In addition to the correctly processed TNFα, some non-specific cleavage was observed, which resulted in a shortened N-terminus. This was still better than in vivo cleavage where only truncated forms of TNFα were obtained. Although the fusion protein was cleaved by enterokinase directly in the medium before purification, this shorter N-terminus was probably a consequence of aberrant enterokinase cleavage. Isolation of fusion protein with His-tag by affinity chromatography with immobilized metal chelate (although normally fast and easy) was not possible because the sequence of five consecutive histidines attached to the N-terminus of the glucoamylase fusion partner was completely cleaved off by proteolysis

Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes

<p>Abstract</p> <p>Background</p> <p>Recombinant protein production in <it>Escherichia coli </it>cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this process. Lately, qPCR is becoming the method of choice for this purpose. In the presented work, an improved qPCR method adopted for PCN determination in various fermentation processes was developed.</p> <p>Results</p> <p>To avoid experimental errors arising from irreproducible DNA isolation, whole cells, treated by heating at 95°C for 10 minutes prior to storage at -20°C, were used as a template source. Relative quantification, taking into account different amplification efficiencies of amplicons for chromosome and plasmid, was used in the PCN calculation. The best reproducibility was achieved when the efficiency estimated for specific amplicon, obtained within one run, was averaged. It was demonstrated that the quantification range of 2 log units (100 to 10000 bacteria per well) enable quantification in each time point during fermentation. The method was applied to study PCN variation in fermentation at 25°C and the correlation between PCN and protein accumulation was established.</p> <p>Conclusion</p> <p>Using whole cells as a template source and relative quantification considering different PCR amplification efficiencies are significant improvements of the qPCR method for PCN determination. Due to the approaches used, the method is suitable for PCN determination in fermentation processes using various media and conditions.</p

Attachment of Histidine Tags to Recombinant Tumor Necrosis Factor-Alpha Drastically Changes Its Properties

When studying two different histidine tags attached to the N-termini of the trimeric cytokine tumor necrosis factor alpha (TNF), the biological activity — measured as cytotoxicity on the L-929 cell line — of both tagged proteins was drastically reduced. The longer His10 tag reduced cytotoxicity to approximately 16% and the shorter His7 tag to 6% of the activity of their nontagged counterparts. After removal of the tags, biological activities reverted to the expected normal values, which clearly shows the key role of the attached histidine tags in diminishing biological activity. Studies on the mechanism of these effects revealed no specific interactions and showed that even the natural flexible N-terminus of TNF presents a steric hindrance for receptor binding, while any extension of the N-terminus increases this hindrance and consequently reduces biological activity. Also, in other proteins, the ligand or substrate binding sites may be hindered by histidine tags, leading to wrong conclusions about biological activity or other properties of the proteins. Thus caution is advised when using His-tagged proteins directly in screening procedures or in research

In vivo and in vitro Cleavage of Glucoamylase-TNFα Fusion Protein Secreted from Aspergillus niger

The most common expression strategy for secreting heterologous proteins from filamentous fungus Aspergillus niger is based on fusion with glucoamylase gene which contains cleavage site for kexin protease (KEX2). However, secretion of recombinant proteins in the form of a fusion-protein without a host-specific cleavage site is usually higher than secretion of the mature protein obtained after in vivo cleavage. We tried to take advantage of such a higher production by cleaving the fusion protein in vitro after fermentation, instead of in vivo during secretion. Similar level of production as after in vivo cleavage was found when human tumor necrosis factor α (TNFα) was produced as a fusion protein with glucoamylase having the enterokinase cleavage site. In addition to the correctly processed TNFα, some non-specific cleavage was observed, which resulted in a shortened N-terminus. This was still better than in vivo cleavage where only truncated forms of TNFα were obtained. Although the fusion protein was cleaved by enterokinase directly in the medium before purification, this shorter N-terminus was probably a consequence of aberrant enterokinase cleavage. Isolation of fusion protein with His-tag by affinity chromatography with immobilized metal chelate (although normally fast and easy) was not possible because the sequence of five consecutive histidines attached to the N-terminus of the glucoamylase fusion partner was completely cleaved off by proteolysis