Genoveva Nacheva

Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli

BackgroundSegregation of expression plasmids leads to loss of recombinant DNA from transformed bacterial cells due to the irregular distribution of plasmids between the daughter cells during cell division. Under non-selective conditions this segregational instability results in a heterogeneous population of cells, where the non-productive plasmid-free cells overgrow the plasmid-bearing cells thus decreasing the yield of recombinant protein. Amongst the factors affecting segregational plasmid instability are: the plasmid design, plasmid copy-number, host cell genotype, fermentation conditions etc. This study aims to investigate the influence of transcription and translation on the segregation of recombinant plasmids designed for constitutive gene expression in Escherichia coli LE392 at glucose-limited continuous cultivation. To this end a series of pBR322-based plasmids carrying a synthetic human interferon-gamma (hIFNγ) gene placed under the control of different regulatory elements (promoter and ribosome-binding sites) were used as a model.ResultsBacterial growth and product formation kinetics of transformed E. coli LE392 cells cultivated continuously were described by a structured kinetic model proposed by Lee et al. (1985). The obtained results demonstrated that both transcription and translation efficiency strongly affected plasmid segregation. The segregation of plasmid having a deleted promoter did not exceed 5% after 190 h of cultivation. The observed high plasmid stability was not related with an increase in the plasmid copy-number. A reverse correlation between the yield of recombinant protein (as modulated by using different ribosome binding sites) and segregational plasmid stability (determined by the above model) was also observed.ConclusionsSwitching-off transcription of the hIFNγ gene has a stabilising effect on ColE1-like plasmids against segregation, which is not associated with an increase in the plasmid copy-number. The increased constitutive gene expression has a negative effect on segregational plasmid stability. A kinetic model proposed by Lee et al. (1985) was appropriate for description of E. coli cell growth and recombinant product formation in chemostat cultivations.

N-terminal methionine in recombinant proteins expressed in two different Escherichia coli strains.

Two genes coding for chloramphenicol acetyltransferase and human interferon gamma, respectively, were overexpressed constitutively in two different strains of Escherichia coli (E. coli LE392 and E. coli XL1). The N-terminal amino acid analysis of the purified proteins showed that: (a) the N-terminal methionine is processed more efficiently in E. coli LE392 rather than in E. coli XL1 cells; (b) the N-terminal methionine is removed better from the heterologous human interferon gamma in comparison with the homologous chloramphenicol acetyltransferase protein: and (c) there is no strong correlation between the efficiency of N-terminal procession and the yield of recombinant protein.

Purification and refolding of recombinant human interferon-gamma in urea-ammonium chloride solution

A method for purification and refolding of recombinant human interferon-gamma (hIFNgamma) from inclusion bodies is described. It includes the following steps: (i) solubilization of inclusion bodies in 7.4 M guanidinium hydrochloride; (ii) purification of the denatured hIFNgamma by hydrophobic chromatography on Octyl-Sepharose column (one step elution with 6 M urea/1 M ammonium chloride); (iii) refolding of the partly purified protein in 0.75 M urea, 20 mM Tris-HCl, pH 8.2; (iv) purification of the refolded protein by CM-Sepharose chromatography. The protein thus obtained is characterized by the following general parameters: yield 1.0 mg/g wet cell mass; purity >99%; specific activity 2x10(8)IU/mg; stability - more than two years as a lyophilized powder and more than two months in solution at 4 degrees C.

Fluorescence Studies on Denaturation and Stability of Recombinant Human Interferon-Gamma

Unfolding/folding transitions of recombinant human interferon-gamma (hIFNγ) in urea and guanidine chloride (Gn.HCl) solutions were studied by fluorescence spectroscopy. At pH 7.4 Gn.HCl was a much more efficient denaturant (midpoint of unfolding C* = 1.1 m and ΔG0 = 13.4 kJ/mol) than urea (C* = 2.8 m and ΔG0 = 11.7 kJ/mol). The close ΔG0 values indicate that the contribution of electrostatic interactions to the stability of hIFNγ is insignificant. Both the pH dependence of the fluorescence intensity and the unfolding experiments in urea at variable pH showed that hIFNγ remains native in the pH range of 4.8-9.5. Using two quenchers, iodide and acrylamide, and applying the Stern-Volmer equation, a cluster of acidic groups situated in close proximity to the single tryptophan residue was identified. Based on the denaturation experiments at different pH values and on our earlier calculations of the electrostatic interactions in hIFNγ, we assume that the protonation of Asp63 causes conformational changes having a substantial impact on the stability of hIFNγ.

Role of the C-terminal chain in human interferonγ stability: An electrostatic study

Electrostatic interactions in two structures of human interferon gamma (hIFNγ), corresponding to interferon molecule alone and bound to its receptor, were analyzed on the basis of a continuum dielectric model. It was found that a number of titratable groups, mainly basic, show large pK shifts and remain in their neutral forms at physiologically relevant pH. The fact that these groups are largely common to both structures and that most of them belong to the set of most conserved sites suggests that this is a property inherent to the hIFNγ molecule rather than an artifact of the crystal packing. His111 was also found deprotonated at neutral pH. It was concluded that receptor recognition involving His111 is driven by aromatic coupling of His111 and Tyr52 from the receptor rather than by electrostatic interactions. The structure corresponding to hIFNγ in complex with its receptor shows a reduction in number and in degree of desolvation of the buried titratable sites. This finding suggested that on receptor binding, hIFNγ adopts energetically more favorable, relaxed, conformation. It was experimentally shown that in contrast to the full‐size hIFNγ, the construct having 21 amino acid residues deleted from the C‐terminus is soluble. The hydrophobicity profile analysis suggested that factors other than the exposure of hydrophobic parts of the molecule are responsible for the low stability and propensity for aggregation. On the basis of these results, it was assumed that the electrostatic influence of the C‐terminal part contributes particularly to the low solvent exposure of the titratable groups, and hence to the low structural stability and propensity for aggregation of the recombinant hIFNγ. Proteins 2001;43:125–133.

SIGNIFICANCE OF THE PUTATIVE UPSTREAM POLYBASIC NUCLEAR LOCALISATION SEQUENCE FOR THE BIOLOGICAL ACTIVITY OF HUMAN INTERFERON-GAMMA

ABSTRACT Interferon-gamma (IFNγ) accomplishes its multiple biological effects by activating STAT transcription factors, which are translocated to the nucleus through a specific nuclear localization sequence(s) (NLS) in the IFNγ molecule. Two putative NLS have been pointed out in the human interferon gamma (hIFNγ): an upstream NLS located in helix E (residues 83–89) and a downstream NLS located at the C-terminal unstructured region (residues 124–132). To investigate the significance of the putative upstream NLS for the biological activity of hIFNγ we have introduced a point mutation in the hIFNγ gene to disturb the polybasic sequence typical for the conventional NLS. In the new gene a Gln codon was substituted for the Lys88 codon and the mutated gene was cloned and expressed in E. coli LE392. This mutation led to a 1000-fold decrease in both hIFNγ antiviral and antiproliferative activities. When co-incubated with the wild-type hIFNγ (standard), the mutant hIFNγ competed for the cellular receptors that led to a 30% inhibition of the standard activity. This indicates that the mutation does not interfere with the interaction of the protein to the receptor but probably affects the intracellular signal transduction pathway. To avoid any putative compensatory effect in the function of the upstream NLS caused by the downstream C-terminal NLS, 21 C-terminal codons were deleted from the mutant hIFNγ gene. The latter resulted in only 10-fold additional decrease in biological activity and 50% inhibition of the standard activity in the competition assay. Our data indicate that the upstream NLS is endowed with a greater functional significance for the hIFNγ mediated signal transduction rather than the downstream NLS.

Expression of Human Interferon-Gamma Gene in Human Tissue Culture Cells

ABSTRACT Interferon-gamma is a cytokine secreted by natural killer cells, CD4 TH1 cells and CD8 cytotoxic lymphocytes in response to mitogenic and antigenic stimuli. It is endowed with multiple biological activities and because of that is one of the first targets of recombinant DNA technology. Human interferon-gamma (hIFNγ) is expressed so far in Escherichia coli and various eukaryotic hosts. The disadvantages of the bacterial hIFNγ and unsatisfactory results from the eukaryotic expression were the reason to employ transient expression of the hIFNγ gene in CAP-T and HEKF cells, widely used for industrial production of human proteins. The obtained results demonstrated that (unlike with other human genes): i) cell viability decreased very fast and reached the critical level of 50% after five days only; ii) cell density did not exceed 0.7x105 cells/mL and sharply decreased after four days of cultivation; iii) the yield of hIFNy secreted in the supernatant did not exceed 5.8 mg/L.

Segregational Instability in E. Coli of Expression Plasmids Carrying Human Interferon Gamma Gene and its 3’-End Truncated Variants

ABSTRACT Segregation of the expression plasmids under non-selective conditions is due to the irregular distribution of the plasmid molecules between the daughter cells during cell division. Thus a heterogeneous population of cells appears in which the non-productive plasmid-free cells overgrow the plasmid-bearing once. The main factors that influence plasmid segregational instability are: plasmid structure, plasmid copy number, host cell genotype, level of protein expression, fermentation conditions, etc. The aim of this work is to investigate the effect of recombinant gene structure and level of expression on the segregation of expression plasmids based on pBR322. To this end the wild type human interferon gamma (hIFNγ) gene and a series of seven derivative genes obtained by systemic 3′-end deletion were inserted into an expression plasmid under a strong constitutive promoter. The segregational instability was studied under batch fermentation conditions in non-selective Luria Bertani medium, where the cell cultures were maintained at an exponential growth phase. To describe the population dynamics, a mathematical model proposed by Stewart & Levin was used and the parameters Δ (difference in specific growth rate between plasmid-free and plasmid-harbouring cells) and θ (relative plasmid loss rate) were determined for each plasmid. The obtained results showed that any variation in the 3′-end of the hIFNγ-gene strongly affected plasmid instability. We also found that the relative plasmid loss rate θ was proportional to the hIFNγ-mRNA content in the cell. Based on these results, we conclude that the structure of the constitutively expressed gene is of great importance for the segregational plasmid instability.

A NEW APPROACH FOR PURIFICATION OF RECOMBINANT HUMAN INTERFERON GAMMA EXPRESSED IN ESCHERICHIA COLI

ABSTRACT A new approach for improving and scaling up the purification of recombinant human interferon gamma (hIFNγ) expressed in Escherichia coli and accumulated in the form of inclusion bodies was investigated. The new strategy involves denaturation of the inclusion bodies in 7.4M guanidine hydrochloride, loading on a hydrophobic (Octyl-Sepharose) column and elution with urea/ammonium chloride. The method is fast, simple and low in cost. For maximum recovery of the hIFNγ biological activity, an optimization of the refolding step is proposed.

In Silico Studies on the Stability of Human Interferon-Gamma Mutants

ABSTRACT Human interferon-gamma (hIFNγ) is a key cytokine in the realisation of cellular immunity. It accomplishes its biological activity upon binding to a specific cell receptor thus inducing the JAK/STAT1 signal transduction pathway. Two putative NLS sequences were pointed out to assist in the translocation of STAT1 into the nucleus. In order to employ mutational analysis for study the biological significance of the polybasic sequence Lys86-Lys87-Lys88 belonging to the upstream putative NLS, hIFNγ mutants with preserved structure and intact binding affinity to cell receptor need to be selected. To this end in silico studies of molecular stability of hIFNγ mutants was performed. The potential conformational changes in the structure of the mutant proteins were investigated employing molecular dynamics simulations. The free energy surface of Lys86 backbone torsion angles space in hIFNγ wild type and mutants was analyzed using metadynamic model. The obtained in silico results were verified by construction of selected mutant recombinant hIFNy proteins, which were analysed for biological activity. To judge for the secondary structure of the mutants the affinity to the cell receptor was investigated. High correlation between results of the molecular dynamics simulations and biological data was obtained.