Gene Expression Profiling of Recombinant Protein Producing E. coli at Suboptimal Growth Temperature.

Recent studies have revealed that at lower cultivation temperatures (25 °C) much higher percentage of correctly folded recombinant hG-CSF protein can be extracted from inclusion bodies. Hence, the goal of our research was to investigate mechanisms determining characteristics of non-classical inclusion bodies production using gene expression profiling, focusing on proteases and chaperones gene expression. Statistical analysis of microarray data showed prominent changes in energy metabolism, in metabolism of amino acids and nucleotides, as well as in biosynthesis of cofactors and secondary metabolites if the culture was grown below its optimal temperature.

Reprint of: Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins.

The new aspect concerning the applicability of histidine and other affinity tags for the purification of oligomeric proteins, with particular emphasis on cleavage efficiency and final yield, is presented in this study. The final yield depends on both the cleavage efficiency and the degree of oligomerization of the protein. Cleavage procedures that are good enough for monomeric proteins can be problematic for oligomeric proteins.

Identification of the heparin-binding domain of TNF-alpha and its use for efficient TNF-alpha purification by heparin-Sepharose affinity chromatography.

The N-terminus of the trimeric TNF-alpha molecule comprises two basic arginines within the short amino-acid sequence VRSSSR, which is here shown to be essential for binding of TNF-alpha to heparin-Sepharose. Mixed trimers containing full-length and DeltaN6-truncated subunits revealed a single VRSSSR sequence to be sufficient to achieve binding. On the basis of this newly identified heparin-binding domain, a new method for efficient purification of TNF-alpha is described.

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

Background: Recombinant protein production in Escherichia coli 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.

Obstacles and pitfalls in the PEGylation of therapeutic proteins.

In recent years, PEGylation has become widely used as a post-production modification methodology for improving the biomedical efficacy and physicochemical properties of therapeutic proteins. Several marketed drugs that have already been in use for more than a decade have proved the applicability and safety of this technology and, with the successes already achieved, it is expected that PEGylation will be applied to other potential therapeutic proteins. The non-biodegradable nature of PEG, however, may become a limiting factor for the next generation of protein pharmaceuticals, for which use in high concentrations and in the long term is the aim, especially considering the trend for the use of branched and high molecular mass PEGs.

New properties of inclusion bodies with implications for biotechnology.

Human G-CSF (granulocyte colony-stimulating factor) is a well-known biopharmaceutical drug being mostly produced by overexpression in Escherichia coli, where it appears in the form of IBs (inclusion bodies). Following our initial findings that properties of inclusion bodies strongly depend on the growth conditions used, especially growth temperature, we compared the characteristics of the G-CSF inclusion bodies prepared at two different temperatures, namely 42 and 25 degrees C. IBs formed at higher growth temperatures have properties similar to the usually described IBs, containing mainly denatured recombinant protein and being almost completely insoluble in aqueous solutions containing mild detergents or low concentrations of denaturants.

Thermal stability of the WHO international standard of interferon alpha 2b (IFN-alpha 2b): application of new reporter gene assay for IFN-alpha 2b potency determinations.

A World Health Organization requirement for biological standards is that they should exhibit long-term stability at their recommended storage temperature. Thermal stability is usually predicted in accelerated thermal degradation studies, where ampoules of the lyophilized standard are stored at elevated temperatures for relatively short times before testing. To confirm the predicted thermal stability of the 2nd international standard of human interferon alpha 2b (IFN-alpha2b; 95/566), we tested the potency of the ampouled contents of this standard after 9 years storage at the customary storage temperature of -20 degrees C in comparison with ampoules of the IS which had been stored continuously at temperatures ranging from -150 degrees C to 56 degrees C.

Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins.

The new aspect concerning the applicability of histidine and other affinity tags for the purification of oligomeric proteins, with particular emphasis on cleavage efficiency and final yield, is presented in this study. The final yield depends on both the cleavage efficiency and the degree of oligomerization of the protein. Cleavage procedures that are good enough for monomeric proteins can be problematic for oligomeric proteins.

Improvement of potential therapeutic value of tumor necrosis-alpha (TNF-alpha) by charge modulation in the tip region.

Analysis of published data reveals that the introduction of more basic amino acid residues in the flexible N-terminal region of the human tumour necrosis factor alpha (TNF) molecule indicates a weak but consistent trend towards increased in vitro cytotoxicity, especially when the effect of N-terminal length is taken into account. In our laboratory, a series of TNF analogues with a charge modification in the tip region of the molecule was prepared, and cytotoxicity measured. Similar trends in cytotoxicity with increasing basicity of the TNF analogue were found in this study for two mouse cell lines, L929 and WEHI-164 clone 13-1, as well as for the human line KYM-1D4.

Production of nonclassical inclusion bodies from which correctly folded protein can be extracted.

Human granulocyte-colony stimulating factor (hG-CSF), an important biopharmaceutical drug used in oncology, is currently produced mainly in Escherichia coli. Expression of human hG-CSF gene in E. coli is very low, and therefore a semisynthetic, codon-optimized hG-CSF gene was designed and subcloned into pET expression plasmids.

High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles.

Wild type gene for green fluorescent protein (GFP) was stably integrated into the Pichia pastoris genome and yielded an expression level of over 40% of total cellular protein. The high cytoplasmic concentration of fluorescent (properly folded and processed) GFP caused the formation of fluorescent spherical structures, which could be observed by fluorescence or confocal microscopy after controlled permeabilization of the yeast cells with 0.2% N-lauroyl sarcosine (NLS).

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.

Constitutive versus thermoinducible expression of heterologous proteins in Escherichia coli based on strong PR,PL promoters from phage lambda.

Constitutive and thermoinducible expression plasmids based on strong P(R),P(L) promoters from phage lambda were compared for production of TNF-alpha and its analogs under various conditions. Much higher accumulation of TNF was obtained in a constitutive system, so the wider applicability of such systems was studied. In constitutive systems, proteolytically susceptible proteins can be produced easily at low cultivation temperatures and the addition of expensive or toxic chemical inducers is not required.

Increased in vitro cytotoxicity of TNF-alpha analog LK-805 is based on the interaction with cell surface heparan sulfate proteoglycan.

Our tumor necrosis factor-alpha (TNF-alpha) analog LK-805 (E107K) exhibited twofold higher specific cytotoxicity on the mouse fibroblast L-929 cell line than its native counterpart. In addition, significantly lowered systemic toxicity was observed in tumor-bearing mouse models treated with this analog. Due to a charge reversal and clustering of three lysines in the exposed tip region of LK-805, we assumed that additional ionic interactions between the positively charged TNF analog and the negatively charged components of the cell surface were created, which might contribute to improved properties of LK-805.

Early events in TNFa - p55 receptor interactions-experiments with TNF dimers.

The first essential step in TNF signal transduction is believed to be clustering of the membrane bound receptors around the trimeric TNF molecule. To check if one receptor binding site would be enough to trigger the signal, we tried to prepare several types of TNF dimer. For this purpose, two TNF analogs bearing different cysteine mutations at the inner subunit binding surfaces were designed, expressed in E.

Soluble tumor necrosis factor receptor I (sTNFRI) as a prognostic factor in melanoma patients in Slovene population.

Tumor necrosis factor α (TNF-α) and its receptors (TNFRI and TNFRII) which exist in soluble form as a product of cleavage of the extracellular domain of membrane integrated receptors, still rise debate about their importance. It was reported that TNF-α has numerous actions in diseases such as inflammation, autoimmunity, infectious diseases, septic shock and many types of cancer [1, 2]. Several authors have reported the significance of sTNFRI level in serum of cancer patients [3, 4].