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1.
Microb Cell Fact ; 18(1): 190, 2019 Nov 05.
Article in English | MEDLINE | ID: mdl-31690339

ABSTRACT

BACKGROUND: The focus of this study is online estimation of biomass concentration in fed-batch cultures. It describes a bioengineering software solution, which is explored for Escherichia coli and Saccharomyces cerevisiae fed-batch cultures. The experimental investigation of both cultures presents experimental validation results since the start of the bioprocess, i.e. since the injection of inoculant solution into bioreactor. In total, four strains were analyzed, and 21 experiments were performed under varying bioprocess conditions, out of which 7 experiments were carried out with dosed substrate feeding. Development of the microorganisms' culture invariant generic estimator of biomass concentration was the main goal of this research. RESULTS: The results show that stoichiometric parameters provide acceptable knowledge on the state of biomass concentrations during the whole cultivation process, including the exponential growth phase of both E. coli and S. cerevisiae cultures. The cell culture stoichiometric parameters are estimated by a procedure based on the Luedeking/Piret-model and maximization of entropy. The main input signal of the approach is cumulative oxygen uptake rate at fed-batch cultivation processes. The developed noninvasive biomass estimation procedure was intentionally made to not depend on the selection of corresponding bioprocess/bioreactor parameters. CONCLUSIONS: The precision errors, since the bioprocess start, when inoculant was injected to a bioreactor, confirmed that the approach is relevant for online biomass state estimation. This included the lag and exponential growth phases for both E. coli and S. cerevisiae. The suggested estimation procedure is identical for both cultures. This approach improves the precision achieved by other authors without compromising the simplicity of the implementation. Moreover, the suggested approach is a candidate method to be the microorganisms' culture invariant approach. It does not depend on any numeric initial optimization conditions, it does not require any of bioreactor parameters. No numeric stability issues of convergence occurred during multiple performance tests. All this makes this approach a potential candidate for industrial tasks with adaptive feeding control or automatic inoculations when substrate feeding profile and bioreactor parameters are not provided.


Subject(s)
Batch Cell Culture Techniques/methods , Escherichia coli/growth & development , Oxygen/metabolism , Saccharomyces cerevisiae/growth & development , Fermentation , Oxygen Consumption
2.
Biochimie ; 91(9): 1123-30, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19524011

ABSTRACT

In this study the bioactivity of three differently glycosylated blood coagulation factor VII (FVII) variants (human plasma FVII, recombinant human FVII produced in CHO and BHK cell cultures) were analyzed and compared. Surface plasmon resonance studies of FVII interaction with soluble and full length TF together with FVII autoactivation assays revealed that BHK-derived FVII has the highest bioactivity, while human plasma and CHO-derived FVII showed very similar bioactivity. The affinity of FVII variants to TF correlates with FVII autoactivation rates--the higher the affinity, the faster the autoactivation rate.


Subject(s)
Factor VII/metabolism , Recombinant Proteins/metabolism , Animals , CHO Cells , Cell Line , Chromatography, High Pressure Liquid , Cricetinae , Cricetulus , Factor VII/genetics , Glycosylation , Humans , Kinetics , Protein Binding , Recombinant Proteins/genetics , Surface Plasmon Resonance , Thromboplastin/metabolism
3.
Int J Biol Macromol ; 44(5): 428-34, 2009 Jun 01.
Article in English | MEDLINE | ID: mdl-19428477

ABSTRACT

Cyclodextrins with different ring size and ring substituents were tested for recombinant mink and porcine growth hormones aggregation suppression in the refolding process from Escherichia coli inclusion bodies. Methyl-beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin show a positive effect on the aggregation suppression of both proteins. The influence of different methyl-beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin concentrations on the renaturation yield of both growth hormones was investigated. Moreover, methyl-beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin suppress not only folding-related, but also temperature-related aggregates formation of both proteins. Circular dichroism experiments (monitoring of protein solution turbidity by registering high tension voltage) showed that the onset temperature of aggregation of both growth hormones increased with increasing 2-hydroxypropyl-beta-cyclodextrin concentration. In conclusion, cyclodextrins have perspectives in biotechnology of veterinary growth hormones not only for protein production, but also for its storage.


Subject(s)
Cyclodextrins/pharmacology , Escherichia coli/genetics , Growth Hormone/chemistry , Growth Hormone/metabolism , Inclusion Bodies/metabolism , Protein Renaturation/drug effects , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Animals , Chromatography, High Pressure Liquid , Circular Dichroism , Cyclodextrins/chemistry , Dose-Response Relationship, Drug , Escherichia coli/cytology , Growth Hormone/genetics , Inclusion Bodies/chemistry , Mink , Protein Binding/drug effects , Protein Folding/drug effects , Recombinant Proteins/genetics , Solubility , Swine , Temperature
4.
Biophys J ; 95(7): 3222-31, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18599640

ABSTRACT

Equilibrium binding ligands usually increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. High-throughput screening for the discovery of drug-like compounds uses an assay based on thermal stabilization. The mathematical description of this stabilization is well developed, and the method is widely applicable to the characterization of ligand-protein binding equilibrium. However, numerous cases have been experimentally observed where equilibrium binding ligands destabilize proteins, i.e., diminish protein melting temperature by an amount proportional to the concentration and affinity of the ligand. Here, we present a thermodynamic model that describes ligand binding to the native and unfolded (denatured) protein states explaining the combined stabilization and destabilization effects. The model also explains nonsaturation and saturation effects on the protein melting temperature when the ligand concentration significantly exceeds the protein concentration. Several examples of the applicability of the model are presented, including specific sulfonamide binding to recombinant hCAII, peptide and ANS binding to the Polo-box domain of Plk1, and zinc ion binding to the recombinant porcine growth hormone. The same ligands may stabilize and destabilize different proteins, and the same proteins may be stabilized and destabilized by different ligands.


Subject(s)
Models, Molecular , Proteins/chemistry , Proteins/metabolism , Temperature , Animals , Carbonic Anhydrases/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Dose-Response Relationship, Drug , Growth Hormone/metabolism , Hot Temperature , Humans , Ligands , Protein Binding , Protein Denaturation/drug effects , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , Quantum Theory , Swine/metabolism , Thermodynamics , Transition Temperature/drug effects , Zinc/metabolism , Zinc/pharmacology , Polo-Like Kinase 1
5.
Biomed Chromatogr ; 22(9): 1001-7, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18506902

ABSTRACT

The formation of the complexes between Cibacron blue F3G-A and two therapeutic proteins, recombinant human interferon-alpha2b and recombinant human growth hormone, was investigated. The method of time-resolved limited proteolysis coupled with MALDI-TOF mass spectrometry was used. The analysis of peptide maps revealed that A(17)HR(19) and L(20)HQLAFDTYQEFEEAYIPK(38) of hGH, and R(14)TLMLLAQMR(23) and D(33)RHDFGFPQEEFGNQFQK(50) of hIFN-alpha2b, exhibit affinity to Cibacron blue F3G-A.


Subject(s)
Coloring Agents/chemistry , Human Growth Hormone/chemistry , Interferon-alpha/chemistry , Peptides/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Triazines/chemistry , Drug Interactions , Humans , Interferon alpha-2 , Ligands , Recombinant Proteins
6.
Protein J ; 27(3): 170-80, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18080174

ABSTRACT

Fourier-transform infrared spectroscopy, in vitro bioassay and enzyme-linked immunoassay were used to study the structural-functional relationships of recombinant mink growth hormone (mGH), refolded and stored under different conditions. Porcine GH (pGH) was synthesized and used as an example. These two hormones, when refolded and stored the same way, had the same secondary structures, biological and immunological efficacy, and biological potency. Only the immunological potency differed, mGH being significantly less potent than pGH. Renaturation pH and storing frozen or at 4 degrees C in 5% glycerol did not affect either the secondary structure or the activity. However, freeze-drying raised the content of buried alpha-helices and lowered that of solvated alpha-helices and of unordered structures. These conformational changes were associated with a reduction of immunological and biological potency of mGH and of immunological potency of pGH. These findings provide original information on the secondary structure of mGH, and show that conformational changes induced by lyophilization adversely affect its activity.


Subject(s)
Growth Hormone/chemistry , Growth Hormone/immunology , Mink/immunology , Protein Renaturation , Amino Acid Sequence , Animals , Cell Line, Tumor , Freeze Drying , Growth Hormone/genetics , Mice , Mink/genetics , Molecular Sequence Data , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sequence Alignment , Spectroscopy, Fourier Transform Infrared , Structure-Activity Relationship , Swine , Temperature
7.
Protein J ; 26(8): 547-55, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17823856

ABSTRACT

L-Arginine was used to suppress the aggregation of recombinant mink and porcine growth hormones in the refolding process from E. coli inclusion bodies by solubilization-dilution protocol at high protein concentration and pH 8.0. The influence of L-arginine concentration on the renaturation yield of both proteins was investigated. L-Arginine effectively suppressed the precipitation of growth hormones during dilution, but did not inhibit soluble oligomers formation. The results of mink and porcine growth hormones purification from 4 g of biomass are presented.


Subject(s)
Arginine/pharmacology , Escherichia coli/genetics , Growth Hormone/metabolism , Inclusion Bodies/metabolism , Protein Denaturation , Protein Folding , Animals , Chromatography, Gel , Chromatography, High Pressure Liquid , Mink , Solubility , Swine
8.
J Chromatogr B Analyt Technol Biomed Life Sci ; 852(1-2): 409-19, 2007 Jun 01.
Article in English | MEDLINE | ID: mdl-17339136

ABSTRACT

High-level expression of recombinant proteins in Escherichia coli frequently leads to the formation of insoluble protein aggregates, termed inclusion bodies. In order to recover a native protein from inclusion bodies, various protein refolding techniques have been developed. Column-based refolding methods and refolding in aqueous two-phase systems are often an attractive alternative to dilution refolding due to simultaneous purification and improved refolding yields. In this work, the effect of surface histidine mutations and their number on the partitioning and refolding of recombinant human granulocyte-colony stimulating factor Cys17Ser variant (rhG-CSF (C17S)) from solubilized inclusion bodies in aqueous two-phase systems polyethylene glycol (PEG)-dextran, containing metal ions, chelated by dye Light Resistant Yellow 2KT (LR Yellow 2KT)-PEG derivative, was investigated. Human G-CSF is a growth factor that regulates the production of mature neutrophilic granulocytes from the precursor cells. Initially, the role of His156 and His170 residues in the interaction of rhG-CSF (C17S) with Cu(II), Ni(II) and Hg(II) ions, chelated by LR Yellow 2KT-PEG, was investigated at pH 7.0 by means of affinity partitioning of purified, correctly folded rhG-CSF (C17S) mutants. It was determined that both His156 and His170 mutations reduced the affinity of rhG-CSF (C17S) for chelated Cu(II) ions at pH 7.0. His170 mutation significantly reduced the affinity of protein for chelated Ni(II) ions. However, histidine mutations had only a small effect on the affinity of protein for Hg(II) ions. The influence of His156 and His170 mutations on the refolding of rhG-CSF (C17S) from solubilized inclusion bodies in aqueous two-phase systems PEG-dextran, containing chelated Ni(II) and Hg(II) ions, was investigated. Reversible interaction of protein mutants with chelated metal ions was used for refolding in aqueous two-phase systems. Both histidine mutations resulted in a significant decrease of protein refolding efficiency in two-phase systems containing chelated Ni(II) ions, while in the presence of chelated Hg(II) ions their effect on protein refolding was negligible. Refolding studies of rhG-CSF variants with different number of histidine mutations revealed that a direct correlation exists between the number of surface histidine residues and refolding efficiency of rhG-CSF variant in two-phase systems containing chelated Ni(II) ions. This method of protein refolding in aqueous two-phase systems containing chelated metal ions should be applicable to other recombinant proteins that contain accessible histidine residues.


Subject(s)
Chelating Agents/chemistry , Cysteine/chemistry , Granulocyte Colony-Stimulating Factor/metabolism , Histidine/genetics , Metals/chemistry , Mutation , Serine/chemistry , Animals , Cell Line, Tumor , Electrophoresis, Polyacrylamide Gel , Granulocyte Colony-Stimulating Factor/chemistry , Granulocyte Colony-Stimulating Factor/genetics , Granulocyte Colony-Stimulating Factor/isolation & purification , Mice , Protein Folding , Recombinant Proteins , Water
9.
Appl Microbiol Biotechnol ; 74(2): 316-23, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17103160

ABSTRACT

Escherichia coli cells expressing mink (Mustela vison) growth hormone were grown in a batch fermentation process. The expression level was estimated to be 27% of the total cellular protein after 3 h of induction with 1 mM isopropyl beta-D-thiogalactoside (IPTG). If the expression of mink growth hormone (mGH) was induced with 0.2 mM IPTG, the concentration of target protein was slightly lower and was found to be 23% at the same time after induction. mGH expressed as inclusion bodies was solubilized in 8 M urea and renatured by dilution protocol at a protein concentration of 1.4-2.1 mg/ml in the presence of glutathione pair in a final concentration of 11.3 mM. [GSH]/[GSSG] ratio equal to 2/1 was used. Two-step purification process comprising of ion-exchange chromatography on Q-Sepharose and hydrophobic chromatography on Phenyl-Sepharose was developed. Some 25-30 mg of highly purified and biologically active mGH was obtained from 4 g of biomass. The method presented in this study allows producing large quantities of mGH and considering initiation of scientific investigation on mGH effect on mink in vivo and availability in fur industry.


Subject(s)
Biotechnology/methods , Escherichia coli/metabolism , Growth Hormone/metabolism , Mink/metabolism , Recombinant Proteins/metabolism , Animals , Cell Line , Culture Media , Escherichia coli/genetics , Fermentation , Gene Expression Regulation, Bacterial , Growth Hormone/chemistry , Growth Hormone/genetics , Growth Hormone/isolation & purification , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
10.
Protein J ; 25(6): 369-78, 2006 Sep.
Article in English | MEDLINE | ID: mdl-17024566

ABSTRACT

About 14 proteins were tested for specific oxidative scission catalyzed by metal ions in the presence of ascorbate and oxidizing agents (O(2) or hydrogen peroxide). Only four of them were degraded by Fe(3+)/Fe(2+)- ascorbate, twelve - by Cu(2+)/Cu(+)-ascorbate and two proteins (alpha- and beta-caseins) were degraded by Pd(2+) ions. The rate and the intensity of degradation are very different for various proteins. For the most of tested proteins only a small fraction of molecules was degraded. None of them was degraded completely. Two possible reasons of protein stability against oxidative degradation may be proposed as follows: either there is no metal binding site in a protein molecule, or metal binding ligands of protein undergo a rapid oxidative modification and the metal ion is released from the binding site. Human growth hormone was cut specifically at two sites by Cu(2+)/Cu(+)-ascorbate system. At least one of amino acid residues of this protein was modified by formation of reactive carbonyl.


Subject(s)
Ascorbic Acid/chemistry , Cations/chemistry , Proteins/chemistry , Binding Sites , Electrophoresis, Polyacrylamide Gel , Human Growth Hormone/chemistry , Human Growth Hormone/metabolism , Humans , Hydrogen-Ion Concentration , Molecular Structure , Protein Binding , Proteins/metabolism , Temperature
11.
Acta Biochim Pol ; 53(1): 87-92, 2006.
Article in English | MEDLINE | ID: mdl-16565748

ABSTRACT

The ability of Congo red to form complexes with alpha-proteins, human growth hormone and human interferon-alpha2b, was found by absorption difference spectroscopy. A human growth hormone-Congo red complex was isolated by gel-permeation chromatography, and its visible absorption spectrum was registered in comparison to free dye. The ability of Congo red to induce dimerization of human growth hormone was demonstrated using chemical cross-linking agents 1,3,5-triacryloyl-hexahydro-s-triazine and ethylene glycol bis(succinimidylsuccinate).


Subject(s)
Congo Red/pharmacology , Fluorescent Dyes/pharmacology , Human Growth Hormone/chemistry , Interferon-alpha/chemistry , Biophysics/methods , Chromatography, Gel , Cross-Linking Reagents/pharmacology , Dimerization , Humans , Hydrogen-Ion Concentration , Interferon alpha-2 , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Proteins , Spectrophotometry , Succinimides/chemistry , Triazines/pharmacology
12.
Biomed Chromatogr ; 20(2): 195-9, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16161178

ABSTRACT

The interaction of Cibacron blue F3G-A with two therapeutic proteins, recombinant human growth hormone and recombinant human interferon-alpha2b, has been examined by applying gel-permeation chromatography in combination with the absorption difference spectroscopy. The complexes of these proteins with Cibacron blue F3G-A have been isolated, and their absorbance spectra have been registered. The influence of Cibacron blue F3G-A on the oligomeric state of proteins has been investigated. It was found that Cibacron blue F3G-A promotes the generation of interferon-alpha2b dimers at pH 5.0.


Subject(s)
Chromatography, Gel/methods , Human Growth Hormone/chemistry , Interferon-alpha/chemistry , Triazines/chemistry , Electrophoresis, Polyacrylamide Gel , Humans , Hydrogen-Ion Concentration , Interferon alpha-2 , Molecular Weight , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Spectrophotometry
13.
Eur J Haematol ; 74(3): 241-9, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15693794

ABSTRACT

To obtain topological information about human phagocyte flavocytochrome b558 (Cytb), rabbit anti-peptide antibodies were raised against synthetic peptides mimicking gp91(phox) regions: 1-9 (MGN), 30-44 (YRV), 150-159 (ESY), 156-166 (ARK), 247-257 (KIS-1, KIS-2). Following affinity purification on immobilized peptide matrices, all antibodies but not prebleed controls recognized purified detergent-solubilized Cytb by enzyme-linked immunosorbent assay (ELISA). Affinity-purified antibodies recognizing KIS, ARK and ESY but not YRV, MGN or prebleed IgG specifically detected gp91(phox) in immunoblot analysis. Antibodies recognizing MGN, ESY, ARK and KIS but not YRV or the prebleed IgG fraction labeled intact normal neutrophils. Surprisingly, all antibodies, with the exception of YRV and pre-immune IgG controls, bound both normal and Cytb-negative neutrophils from the obligate heterozygous mother of a patient with X-linked chronic granulomatous disease (X-CGD) and all neutrophils from another patient lacking the gp91(phox) gene. Further immunochemical examination of membrane fractions derived from nine genetically unrelated patients with X-CGD, using an antibody that recognizes other Nox protein family members, suggests that the unusual reactivity observed does not reflect the compensatory expression of gp91(phox) homologs Nox1, 3 or 4. These results suggest that an unusual surface reactivity exists on neutrophils derived from X-linked chronic granulomatous disease patients that most likely extends to normal neutrophils as well. The study highlights the need for caution in interpreting the binding of rabbit polyclonal antipeptide antibodies to human neutrophils in general and, in the specific case of antibodies directed against Cytb, the need for Cytb-negative controls.


Subject(s)
Antigen-Antibody Reactions , Granulomatous Disease, Chronic/immunology , Membrane Glycoproteins/immunology , NADPH Oxidases/biosynthesis , NADPH Oxidases/immunology , Neutrophils/immunology , Amino Acid Sequence , Antibodies/immunology , Cytochrome b Group/immunology , Epitopes , Female , Heterozygote , Humans , Male , Membrane Proteins/analysis , Membrane Proteins/biosynthesis , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/analysis , Peptides/immunology
14.
J Chromatogr B Analyt Technol Biomed Life Sci ; 786(1-2): 17-32, 2003 Mar 25.
Article in English | MEDLINE | ID: mdl-12650998

ABSTRACT

Site directed mutagenesis of Cys17-->Ser17 form of recombinant human granulocyte colony stimulating factor (rhG-CSF C17S) for sequential replacing of surface His(43) and His(52) with alanine was used to identify residues critical for the protein interaction with metal ions, in particular Ni(2+) chelated by dye Light Resistant Yellow 2 KT (LR Yellow 2KT)-polyethyleneglycol (PEG), and refolding after partitioning of inclusion bodies in aqueous two-phase systems. Strong binding of rhG-CSF (C17S) to PEG-LR Yellow 2KT-Cu(II) complex allowed for the adoption of affinity chromatography on Sepharose-LR Yellow 2KT-Cu(II) that appeared to be essential for the rapid isolation of mutated forms of rhG-CSF. Efficiency of that purification stage is exemplified by isolation of rhG-CSF (C17S, H43A) and rhG-CSF (C17S, H43A, H52A) mutants in correctly folded and highly purified state. Affinity partitioning of rhG-CSF histidine mutants was studied in aqueous two-phase systems containing Cu(II), Ni(II) and Hg(II) chelated by LR Yellow 2KT-PEG at pH 7.0 and Cu(II)-at pH 5.0. It was determined, that affinity of rhG-CSF mutants for metal ions decreased in the order of C17S>C17S, H43A>C17S, H43A, H52A for Cu(II), and C17S=C17S, H43A>C17S, H43A, H52A for Ni(II) ions, while affinity of all rhG-CSF mutants for Hg(II) ions was of the same order of magnitude. Influence of His(43) and His(52) mutation on protein refolding was studied by partitioning of the respective inclusion body extract in aqueous two-phase systems containing Ni(II) and Hg(II) ions. Data on rhG-CSF histidine mutant partitioning and refolding indicated, that His(52) mutation is crucial for the strength of protein interaction with chelated Ni(II) ions and refolding efficiency.


Subject(s)
Chelating Agents/metabolism , Granulocyte Colony-Stimulating Factor/metabolism , Histidine/genetics , Recombinant Proteins/metabolism , Chromatography, Affinity , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Granulocyte Colony-Stimulating Factor/chemistry , Granulocyte Colony-Stimulating Factor/genetics , Histidine/chemistry , Humans , Hydrogen-Ion Concentration , Imidazoles/chemistry , Isoelectric Focusing , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Recombinant Proteins/genetics
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