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. Moreover, 24 differentially expressed up to now known genes classified among proteases, chaperones and other heat or stress related genes. Among chaperones UspE and among proteases YaeL and YeaZ might play an important role in accumulation of correctly folded recombinant proteins. Membrane localized protease yaeL gene was found to have higher activity at 25 °C and is thus potentially functionally related to the more efficient recombinant protein production at lower temperatures. The results of this study represent advance in the understanding of recombinant protein production in E. coli. Genes potentially influencing production of recombinant protein at lower growth temperature represent basis for further research towards improvement of E. coli production strains as well as fermentation process.

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. Random distribution of uncleaved or partially cleaved affinity tags among oligomers is the main cause of reduced yields. A trimeric protein, tumour necrosis factor alpha (TNF-alpha), bearing different histidine tags, was used as a model protein to explore and confirm this theoretical concept. Analysis of mixed TNF trimers, prepared from tag-free TNF doped with various amounts of histidine-tagged TNF, revealed an increased retention of the trimeric protein on immobilized metal-ion affinity chromatography (IMAC) columns. When 20% of histidine-tagged TNF was added, more than 50% of the protein was retained on the IMAC column. Thus, the applicability of histidine- and other affinity tags for purifying oligomeric proteins is significantly prejudiced in the case of higher oligomers. Various histidine-tags were fused to the N-terminus of full-length TNF-alpha and to the truncated form (dN6) of TNF-alpha. Two-step IMAC separation was used for purification. In the first step, IMAC-1, over 95% purity of histidine-tagged protein was achieved in all cases. Endo- and exoproteolytic removal of histidine tags with enterokinase (EKmax) and aminodipeptidase (DAPase) was studied and the major parameters affecting cleavage efficiency, microheterogeneity and final yield are critically discussed. IMAC-2 was used as the second and final step for removing the cleavage enzyme, cleaved tags, unprocessed protein and some other impurities. Selection of the optimal cleavage enzyme depends on the amino acid composition of the N-terminus and the intended use of the purified protein. The main conclusion is that special caution should be taken when introducing affinity tags to oligomeric proteins, with the final goal to produce pure, tag-free protein with acceptable yields. Given the same enzyme cleavage efficiency one can expect progressively reduced final protein yields with increasing degree of oligomerization. This should be considered as a general rule.

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. Affinity chromatography on heparin-Sepharose was introduced as a key step for highly purified TNF-alpha at a high yield. With minor modifications, this procedure can be used for TNF-alpha analogues that have full-length N-termini, as shown for the less toxic analogue LK-805.

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. In the presented work, an improved qPCR method adopted for PCN determination in various fermentation processes was developed. RESULTS: To avoid experimental errors arising from irreproducible DNA isolation, whole cells, treated by heating at 95 degrees C for 10 minutes prior to storage at -20 degrees 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 degrees C and the correlation between PCN and protein accumulation was established. CONCLUSION: 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.

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. This review addresses various obstacles and pitfalls in the production of PEGylated biopharmaceuticals, in particular, the specificity of PEGylation reactions, separation and purification issues, the analysis of inherently polydisperse PEG reagents and PEGylated products, the consistency of products and processes, and the accurate determination of pharmacokinetic properties.

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. In contrast, when produced at lower growth temperature of 25 degrees C, IBs show significantly different properties. Such IBs contain a significant proportion of G-CSF that is easily and directly extractable in the biologically active form, using non-denaturing solutions, which can be exploited for environmentally friendly biotechnological production. Irrespective of the production temperature, a significant decrease in IB volume was observed when transferring IBs from neutral to acidic (around 4) pH. Irreversible contraction of IBs at low pH was documented at the macro- and micro-scopic level using electron microscopy as a characterization tool. Together with volume decrease, a higher density, and thus decreased solubility, of IBs was observed at low pH, resulting in slower and less efficient extractability of the target protein.

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. Since IFN-alpha2b potency estimates derived from the results of antiviral assays (AVA) showed high within-assay variability, we investigated a novel reporter gene assay (RGA) based on induction of secreted alkaline phosphatase (SEAP) for comparability and precision of such estimations. We show that this RGA generated comparable estimates with overall lower variation. Additionally, the SEAP conversion of p-nitrophenyl phosphate to yellow product could be followed kinetically. Absorbance readings were shown to increase with time in proportion with increasing concentration of IFN-alpha2b. When the time-dependent increments of absorbance were plotted graphically, the slopes of lines corresponded to concentration. This approach enabled single dilutions of IFN samples, identical in molecular structure to an IFN-alpha2b standard, to be used for potency estimates by interpolation of slope value against those of the standard at fixed concentrations. It appears attractive for high through-put potency testing of various R&D IFN-alpha2b samples.

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. Random distribution of uncleaved or partially cleaved affinity tags among oligomers is the main cause of reduced yields. A trimeric protein, tumour necrosis factor alpha (TNF-alpha), bearing different histidine tags, was used as a model protein to explore and confirm this theoretical concept. Analysis of mixed TNF trimers, prepared from tag-free TNF doped with various amounts of histidine-tagged TNF, revealed an increased retention of the trimeric protein on immobilized metal-ion affinity chromatography (IMAC) columns. When 20% of histidine-tagged TNF was added, more than 50% of the protein was retained on the IMAC column. Thus, the applicability of histidine- and other affinity tags for purifying oligomeric proteins is significantly prejudiced in the case of higher oligomers. Various histidine-tags were fused to the N-terminus of full-length TNF-alpha and to the truncated form (dN6) of TNF-alpha. Two-step IMAC separation was used for purification. In the first step, IMAC-1, over 95% purity of histidine-tagged protein was achieved in all cases. Endo- and exoproteolytic removal of histidine tags with enterokinase (EKmax) and aminodipeptidase (DAPase) was studied and the major parameters affecting cleavage efficiency, microheterogeneity and final yield are critically discussed. IMAC-2 was used as the second and final step for removing the cleavage enzyme, cleaved tags, unprocessed protein and some other impurities. Selection of the optimal cleavage enzyme depends on the amino acid composition of the N-terminus and the intended use of the purified protein. The main conclusion is that special caution should be taken when introducing affinity tags to oligomeric proteins, with the final goal to produce pure, tag-free protein with acceptable yields. Given the same enzyme cleavage efficiency one can expect progressively reduced final protein yields with increasing degree of oligomerization. This should be considered as a general rule.

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. For the series of analogues as a whole, a general increase in in vitro cytotoxicity with increasing pI values was not apparent, but some analogues with charge reversal in the tip region, for example, the LK-805 analogue (E107K), exhibited significantly increased cytotoxicity in comparison to native TNF in a range of cell lines, including L929, KYM-1D4-K, WEHI-164 clone 13-1, HEPA 1-6 and EAhy926 cell lines. Experiments with heparinase-pre-treated cells demonstrated that the increased in vitro cytotoxicity of LK-805 is most probably due to interactions with cell surface heparan sulphates that effectively concentrate it before binding to TNF receptors occurs. Examination of structural models of TNF bound to soluble TNF receptor 1 (TNFR1) indicates that simple mutations in the tip region most probably cannot interact with receptor binding sites, and therefore do not directly modulate cytotoxicity.

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. This led to a yield of over 50% of the total cellular proteins. We designed a new approach to biosynthesis at low temperature, enabling the formation of "nonclassical" inclusion bodies from which correctly folded protein can be readily extracted by nondenaturing solvents, such as mild detergents or low concentrations of polar solvents such as DMSO and nondetergent sulfobetaines. FT-IR analysis confirmed different nature of inclusion bodies with respect to the growth temperature and indicated presence of high amounts of very likely correctly folded reduced hG-CSF in nonclassical inclusion bodies. The yield of correctly folded, functional hG-CSF obtained in this way exceeded 40% of the total hG-CSF produced in the cells and is almost completely extractable under nondenaturing conditions. The absence of the need to include a denaturation/renaturation step in the purification process allows the development of more efficient processes characterized by higher yields and lower costs and involving environment-friendly technologies. The technology presented works successfully at the 50-L scale, producing nonclassical inclusion bodies of the same quality. The approach developed for the production of hG-CSF could be extended to other proteins; thus, a broader potential for industrial exploitation is envisaged.

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). Fluorescent GFP particles were also isolated after removal of the cell wall and found to be quite resistant to 0.2% N-lauroyl sarcosine. SDS-PAGE analysis of the isolated fluorescent particles revealed the presence of an 80 kDa protein (alcohol oxidase) and GFP (30%). We conclude that GFP is able to enter spontaneously into the peroxisomes and is inserted into densely packed layers of alcohol oxidase. Consequently, the formation of similar fluorescent particles can also be expected in other organisms when using high-level expression systems. As GFP is widely used in fusion with other proteins as a reporter for protein localization and for many other applications in biotechnology, care must be taken to avoid false interpretations of targeting or trafficking mechanisms inside the cells. In addition, when whole cells or cytoplasmic fractions are used for the quantitative determination of GFP levels, incorrect and misleading values of GFP could be obtained due to the formation of fluorescent particles containing material inside which is not available for fluorescence measurements.

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. On the other hand, toxic proteins cannot be produced and selection pressure must be strictly maintained to ensure segregational stability of plasmids. Accumulation of TNF-alpha and various analogs at levels up to 25% of total soluble protein was repeatedly achieved, which was 2-3-fold higher than in a thermoinducible system. The stable behavior of the constitutive system in laboratory fermentors was also confirmed. We propose the constitutive system described here as a general model for many currently used expression systems containing strong but not completely repressed promoters. Such systems may be considered as constitutive ones with reduced promoter strengths, but still exhibiting all the intrinsic properties of constitutive expression systems. Although all modern expression systems are inducible, wider use of a constitutive system is evidently possible.

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. To prove this hypothesis, we designed truncated forms of TNF-alpha and analog LK-805 and performed three independent sets of experiments: measurement of cytotoxic activity in the presence of excess heparan sulfate, determination of cytotoxic activity on heparinase-treated L-929 cells, and binding of various TNF-alpha proteins onto the heparin-sepharose affinity column. Cytotoxicity studies of both kinds confirmed the pivotal role of the E107K mutation for interaction with heparan sulfate proteoglycans on the cell surface of L-929 cells. However, heparin-binding studies revealed that intact, full-length N-termini of TNF-alpha or its analogs were necessary for high retention on the heparin affinity column, whereas the three-lysine containing tip of LK-805 by itself was not enough for binding. Obviously, immobilized heparin does not represent an adequate model for membrane-bound heparan sulfate proteoglycans of L-929 cells.

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.

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]. This study was performed in collaboration with the Institute of Oncology of Slovenia.At least two different mouse monoclonal antibodies (MAbs) against human sTNFRI have been prepared to obtain a sensitive and reliable sandwich ELISA. It was compared with commercially available R&D and Endogen ELISAs for the determination of sTNFRI. Groups of patients with different stages of melanoma and epithelial ovarian carcinoma were tested and their clinical records were reexamined. Levels of sTNFRI were measured and compared with the normal serum levels of sTNFRI.

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. coli and prepared in pure form. By mixing equimolar quantities of these analogs under appropriate conditions, two different types of dimer were prepared. The first, Dim/S2, proved to be composed mainly of a disulfide-linked dimer, which was still capable of trapping the third subunit of either of the precursor analogs, thus showing relatively high residual cytotoxicity. To avoid trimeric structures, Dim/S2 was further transformed into Dim/Iaa2 by alkylation of -SH groups of the newly introduced cysteines, allowing binding of only two TNF subunits through native contact surfaces. These dimers showed substantially reduced cytotoxicity on the L929 cell line. In addition, it appears that Dim/Iaa2 is able to competitively inhibit cytotoxicity of native TNF, as assessed on the L-M cell line.