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1.
Protein Expr Purif ; 188: 105974, 2021 12.
Article in English | MEDLINE | ID: mdl-34520839

ABSTRACT

Human growth hormone (hGH) plays an important role in growth control, growth promotion, cell development, and regulation of numerous metabolic pathways in the human body and has been approved by the U.S. FDA for the treatment of several human dysfunctions. Over-expression of recombinant hGH (rhGH) affords a misfolded form in cytoplasm of Escherichia coli, and the refolding step required to obtain active rhGH greatly affects its production costs. Herein, the cleavable self-aggregating tag (cSAT) scheme was used for the expression and purification of rhGH in E. coli. Four aggregating tags (L6KD/α3-peptide/EFK8/ELK16) successfully drove rhGH into active protein aggregates. After the Mxe GyrA intein-mediated cleavage, 2.8-21.4 µg rhGH/mg wet cell weight was obtained at laboratory scale, of which the L6KD fusion achieved the highest rhGH yield. The further refined rhGH maintained 92% of the bioactivity compared to commercial rhGH. The self-assembling of the aggregating tag might physically separate the hGH polypeptide chains, which in turn was beneficial to its folding into the active form. This study provided a simple and cost-effective approach for active rhGH production, and suggested an opportunity for improve folding of recombinant proteins in E. coli.


Subject(s)
Gene Expression , Human Growth Hormone/genetics , Inteins/genetics , Recombinant Fusion Proteins/genetics , Amino Acid Sequence , Chromatography, Affinity , Chromatography, Gel , Cloning, Molecular , DNA Gyrase/genetics , DNA Gyrase/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Human Growth Hormone/biosynthesis , Human Growth Hormone/isolation & purification , Humans , Peptides/genetics , Peptides/metabolism , Protein Aggregates , Protein Folding , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification
2.
BMC Biotechnol ; 21(1): 51, 2021 08 16.
Article in English | MEDLINE | ID: mdl-34399745

ABSTRACT

BACKGROUND: Human Growth Hormone (hGH) is a glycoprotein released from the pituitary gland. Due to the wide range of effects in humans, any disruption in hGH secretion could have serious consequences. This highlights the clinical importance of hGH production in the treatment of different diseases associated with a deficiency of this hormone. The production of recombinant mature hormone in suitable hosts and secretion of this therapeutic protein into the extracellular space can be considered as one of the best cost-effective approaches not only to obtain the active form of the protein but also endotoxin-free preparation. Since the natural growth hormone signal peptide is of eukaryotic origin and is not detectable by any of the Escherichia coli secretory systems, including Sec and Tat, and is therefore unable to secrete hGH in the prokaryotic systems, designing a new and efficient signal peptide is essential to direct hGh to the extracellular space. RESULTS: In this study, using a combination of the bioinformatics design and molecular genetics, the protein A signal peptide from Staphylococcus aureus was modified, redesigned and then fused to the mature hGH coding region. The recombinant hGH was then expressed in E. coli and successfully secreted to the medium through the Sec pathway. Secretion of the hGH into the medium was verified using SDS-PAGE and western blot analysis. Recombinant hGH was then expressed in E. coli and successfully secreted into cell culture medium via the Sec pathway. The secretion of hGH into the extracellular medium was confirmed by SDS-PAGE and Western blot analysis. Furthermore, the addition of glycine was shown to improve hGH secretion onto the culture medium. Equations for determining the optimal conditions were also determined. Functional hGH analysis using an ELISA-based method confirmed that the ratio of the active form of secreted hGH to the inactive form in the periplasm is higher than this ratio in the cytoplasm. CONCLUSIONS: Since the native signal protein peptide of S. aureus protein A was not able to deliver hGH to the extracellular space, it was modified using bioinformatics tools and fused to the n-terminal region of hGh to show that the redesigned signal peptide was functional.


Subject(s)
Escherichia coli/genetics , Gene Expression , Human Growth Hormone/genetics , Human Growth Hormone/isolation & purification , Staphylococcal Protein A/genetics , Culture Media/chemistry , Culture Media/metabolism , Escherichia coli/metabolism , Human Growth Hormone/metabolism , Humans , Protein Sorting Signals , Protein Transport , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Staphylococcal Protein A/metabolism
3.
Biotechnol Appl Biochem ; 68(1): 122-135, 2021 Feb.
Article in English | MEDLINE | ID: mdl-32092174

ABSTRACT

Overexpression of insoluble human growth hormone (hGH) in cytoplasm was achieved by E. coli Rosetta-gami B(DE3) [pET21a (+)-hGH]). For overexpression of hGH, effects of eight factors including temperature, type and concentration of carbon source, IPTG and MgSO4 , buffering capacity, induction time, yeast extract/peptone ratio on rhGH production were studied by Plackett-Burman screening. Maximum production of rhGH was 0.681 g/L, and results of statistical analysis showed that induction temperature and glucose have the greatest effect and the presence of MgSO4 increases rhGH expression and reduces biomass concentration. So, the effect of ethanol and MgSO4 concentrations on the rhGH production was examined according to the central composite experimental design. The ANOVA of the results showed rhGH production increases to 1.128 g/L in 4 g/L MgSO4 and 1% ethanol. Then, the impact of glucose concentration and induction time on the rhGH production was evaluated in two levels in the fermenter by Taguchi statistical method. Under optimum conditions, OD600nm 4 and 10 g/L glucose crude rhGH concentration 4.17 g/L was obtained, which is one of the highest value ever reported. Finally, rhGH was purified using the biophysical and biochemical techniques comprising circular dichroism, fluorescent spectroscopy, and dynamic light scattering, and it was confirmed that the produced protein is comparable to the commercial standard sample.


Subject(s)
Escherichia coli , Gene Expression , Human Growth Hormone , Escherichia coli/genetics , Escherichia coli/metabolism , Human Growth Hormone/biosynthesis , Human Growth Hormone/chemistry , Human Growth Hormone/genetics , Human Growth Hormone/isolation & purification , Humans , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
4.
Protein Pept Lett ; 28(5): 533-542, 2021.
Article in English | MEDLINE | ID: mdl-33172365

ABSTRACT

BACKGROUND: Human growth hormone (hGH) is the first recombinant protein approved for the treatment of human growth hormone deficiency. However, expression in inclusion bodies and low expression levels are enormous challenges for heterologous expression of hGH in Escherichia coli. OBJECTIVE: To increase the soluble expression of recombinant hGH with correct folding in E. coli. METHODS: We constructed a new recombinant expression plasmid containing the coding sequence of the outer membrane protein A (ompA3) which was used for the expression in Transetta (DE3) E. coli. In order to simplify the purification process and cleavage of recombinant proteins, the fusion sequence should contain hexahistidine-tag (His6) and enterokinase recognition sites (D4K). The effect of different expression conditions on recombinant hGH expression was optimized in flask cultivations. Furthermore, the periplasmic solution containing soluble hGH was purified by Ni-NTA affinity chromatography. Circular dichroism (CD), western blot and mass spectrometry analyses were used to characterize the protein. Moreover, the growth-promoting effect of the purified hGH was also evaluated by cell proliferation assay. RESULTS: High-level expression (800 µg/mL) was achieved by induction with 0.5 mM IPTG at 30°C for 10 hours. The purity of hGH was over 90%. The immunological activity, secondary structure and molecular weight of the purified hGH were consistent with native hGH. The purified hGH was found to promote the growth of MC3T3-E1 cells, and was found to show the highest activity at a concentration of 100 ng/mL. CONCLUSION: Our research provides a feasible and convenient method for the soluble expression of recombinant hGH in E. coli, and may lay a foundation for the production and application of hGH in the industry.


Subject(s)
Bacterial Outer Membrane Proteins , Escherichia coli Proteins , Escherichia coli , Human Growth Hormone , Recombinant Fusion Proteins , Bacterial Outer Membrane Proteins/biosynthesis , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/isolation & purification , Human Growth Hormone/biosynthesis , Human Growth Hormone/chemistry , Human Growth Hormone/genetics , Human Growth Hormone/isolation & purification , Humans , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification
5.
J Clin Neurosci ; 81: 78-82, 2020 Nov.
Article in English | MEDLINE | ID: mdl-33222975

ABSTRACT

BACKGROUND AND PURPOSE: Recent scientific reports and epidemiological studies have engendered mounting concerns regarding the potential human-to-human transmissibility of non-prion neurodegenerative and related diseases. This study investigated whether recipients of cadaveric pituitary hormone treatments are at increased risk of death from non-prion neurodegenerative and related diseases. METHODS: A retrospective national cohort study based on death certificates of recipients of the cadaveric pituitary hormone treatments (n = 184) as part of the Australian Human Pituitary Hormone Program (AHPHP; n = 2940) 1967-1985. Standardised mortality ratios (SMR) from non-prion neurodegenerative and other diseases were estimated based on the Australian population. RESULTS: Allowing for potential diagnostic mis-attributions, there was no significant increase in the SMR from non-prion central nervous system (CNS) neurodegenerative disease, especially dementia and/or Alzheimer's disease (0.47; [95% CI: 0.19, 1.12] P = 0.081). The SMR for intra-cerebral haemorrhage, potentially related to cerebral amyloid angiopathy (CAA), was increased (2.77; [95% CI: 1.12-5.75] P = 0.009), although accommodation of possible mis-diagnosis through conflation of this category with other stroke causes of death emphasising likely intra-cranial haemorrhage showed no persisting significant increase in mortality in cadaveric pituitary hormone recipients, including all deaths recorded as due to intra-cranial haemorrhage (1.72; [95% CI: 0.80, 3.26] P = 0.123). CONCLUSION: In the setting of recent evidence strongly supporting the likelihood of brain-to-brain horizontal transmission and subsequent propagation and deposition of abnormally folded proteins associated with non-prion neurodegenerative and related disorders, this study offers further tentative support for deaths directly stemming from transmission of non-prion disease related to cadaveric pituitary hormone treatment. Acknowledging the limitations of the present study, however, ongoing detailed assessments of this potential risk are necessary.


Subject(s)
Cerebral Hemorrhage/chemically induced , Cerebral Hemorrhage/mortality , Human Growth Hormone/adverse effects , Neurodegenerative Diseases/chemically induced , Neurodegenerative Diseases/mortality , Adult , Aged , Australia/epidemiology , Brain/drug effects , Brain/pathology , Cadaver , Cerebral Hemorrhage/diagnosis , Cohort Studies , Female , Human Growth Hormone/isolation & purification , Humans , Male , Middle Aged , Neurodegenerative Diseases/diagnosis , Retrospective Studies
6.
Endocrinology ; 161(8)2020 08 01.
Article in English | MEDLINE | ID: mdl-32556100

ABSTRACT

A rare 20K isoform of GH-V (here abbreviated as GHv) was discovered in 1998. To date, only 1 research article has characterized this isoform in vivo, observing that GHv treatment in male high-fat fed rats had several GH-like activities, but unlike GH lacked diabetogenic and lactogenic activities and failed to increase IGF-1 or body length. Therefore, the current study was conducted to further characterize the in vivo activities of GHv in a separate species and in a GH-deficient model (GH-/- mice) and with both sexes represented. GHv-treated GH-/- mice had significant increases to serum IGF-1, femur length, body length, body weight, and lean body mass and reduced body fat mass similar to mice receiving GH treatment. GH treatment increased circulating insulin levels and impaired insulin sensitivity; in contrast, both measures were unchanged in GHv-treated mice. Since GHv lacks prolactin receptor (PRLR) binding activity, we tested the ability of GH and GHv to stimulate the proliferation of human cancer cell lines and found that GHv has a decreased proliferative response in cancers with high PRLR. Our findings demonstrate that GHv can stimulate insulin-like growth factor-1 and subsequent longitudinal body growth in GH-deficient mice similar to GH, but unlike GH, GHv promoted growth without inhibiting insulin action and without promoting the growth of PRLR-positive cancers in vitro. Thus, GHv may represent improvements to current GH therapies especially for individuals at risk for metabolic syndrome or PRLR-positive cancers.


Subject(s)
Growth Hormone/genetics , Human Growth Hormone/pharmacology , Placental Hormones/pharmacology , Animals , Body Composition/drug effects , Body Weight/drug effects , Female , Growth Hormone/deficiency , Hormone Replacement Therapy , Human Growth Hormone/isolation & purification , Human Growth Hormone/metabolism , Human Growth Hormone/therapeutic use , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Placenta/chemistry , Placenta/metabolism , Placental Hormones/therapeutic use , Pregnancy , Protein Isoforms
7.
Talanta ; 198: 330-336, 2019 Jun 01.
Article in English | MEDLINE | ID: mdl-30876569

ABSTRACT

In this study, polyhedral oligomeric silsesquioxane (POSS)-based capillary monoliths with short alkyl chain ligand in the form of butyl (C4) were synthesized via two different polymerization routes, namely UV-initiated free radical copolymerization of methacrylate-derivatized POSS (POSS-MA) with butylmethacrylate (BMA) and UV-initiated thiol-methacrylate copolymerization of POSS-MA with butanethiol (BT). An organosilicon monolith with a pore size distribution lying on both mesoporous and macroporous scales, a lower mean pore size and a higher specific surface area was obtained with UV-initiated thiol-methacrylate polymerization. Both monoliths were then comparatively evaluated for gradient separation of proteins under reversed phase conditions in nano-liquid chromatography. The chromatographic performance was defined in terms of peak-resolution and peak capacity. Four carbon (C4) functionalized-poly(POSS-MA) monolith produced by UV-initiated thiol-methacrylate polymerization exhibited better separation performance with higher peak resolutions and peak capacities. Both, the morphological characterization of monoliths and the results of gradient separation of proteins showed that thiol-methacrylate polymerization was more suitable for the synthesis of C4 functionalized organosilicon based stationary phases for reversed-phase protein separation. The monolith prepared by thiol-methacrylate polymerization was also successfully applied for impurity analysis of two important hormones, namely insulin and genotropin. A comparison with a commercial poly(styrene-co-divinylbenzene) monolith documented the good chromatographic performance of the new BT-attached poly(POSS-MA) monolith.


Subject(s)
Human Growth Hormone/isolation & purification , Insulin/isolation & purification , Nanotechnology , Organosilicon Compounds/chemistry , Chromatography, Liquid , Human Growth Hormone/chemistry , Insulin/chemistry , Methacrylates/chemistry , Organosilicon Compounds/chemical synthesis , Photochemical Processes , Polymerization , Sulfhydryl Compounds/chemistry
8.
Biotechnol Prog ; 34(4): 999-1005, 2018 07.
Article in English | MEDLINE | ID: mdl-29693323

ABSTRACT

Recombinant human growth hormone (rhGH) is used for the treatment of several pathologies, most of them related to growth. Although different expression systems can be used for its production, the milk from transgenic cows is one of the most interesting due to the high rhGH level achieved (5 g/L). We have designed and synthesized short peptides (9 or 10 amino acid long) using Fmoc chemistry and studied their ability to purify rhGH from milk once immobilized on an agarose support. Using spiked milk with the hormone as a sample, rhGH was purified with 88% yield and 92% purity in a single step with a fold purification of 4.5. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:999-1005, 2018.


Subject(s)
Chromatography, Affinity/methods , Human Growth Hormone/isolation & purification , Milk/chemistry , Recombinant Proteins/isolation & purification , Animals , Human Growth Hormone/chemistry , Humans , Protein Array Analysis , Recombinant Proteins/chemistry
9.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1044-1045: 206-213, 2017 Feb 15.
Article in English | MEDLINE | ID: mdl-28153672

ABSTRACT

Human growth hormone plays an essential role in the treatment of dwarfism diseases, but it is limited in its short circulating half-life. Nowadays, some manufacturers are trying to take advantage of polyethylene glycol (PEG) conjugated with recombinant human growth hormone (rhGH) to improve its half-life and efficacy. However, the modified products are heterogeneous mixtures composed of reaction products with different modification sites. It is generally known as a challenging task to separate and characterize a PEGylated product, especially for its positional isoforms. In this study, cation exchange high performance liquid chromatograph (IEC-HPLC) based on a pH gradient separation method was presented to separate five position isomers of rhGH conjugated with a 40-kDa branched PEG N-hydroxysuccinimidyl (NHS) functional group. Then Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALD-TOF MS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that each of five materials collected by IEC-HPLC was conjugated with only one branched PEG chain. Furthermore, rhGH and PEG-rhGH were digested by trypsin and peptides were collected by reversed phase high performance liquid chromatography (RP-HPLC). Following MALDI-TOF MS, PEG modification sites were determined through comparative analysis of peptide mapping between PEG-rhGH and rhGH. Finally, biological activities of those positional isomers were performed in vivo and very small variations were observed. This method was shown to be suitable for heterogeneity analysis of PEGylated biopharmaceutical products.


Subject(s)
Chromatography, Liquid/methods , Human Growth Hormone/analogs & derivatives , Polyethylene Glycols/chemistry , Polyethylene Glycols/isolation & purification , Human Growth Hormone/analysis , Human Growth Hormone/chemistry , Human Growth Hormone/isolation & purification , Human Growth Hormone/metabolism , Humans , Hydrogen-Ion Concentration , Isomerism , Polyethylene Glycols/analysis , Polyethylene Glycols/metabolism
10.
Protein Expr Purif ; 131: 91-100, 2017 03.
Article in English | MEDLINE | ID: mdl-28013084

ABSTRACT

The human growth hormone receptor antagonist G120R-hGH precludes dimerization of GH and prolactin receptors and consequently JAK/STAT signaling. Some modifications in this antagonist resulted in a drug specific for the GH receptor, called Pegvisomant (Somavert®). However, the original G120R-hGH is usually synthesized in bacterial cytoplasm as inclusion bodies, not being a commercial product. The present work describes the synthesis and characterization of G120R-hGH secreted into bacterial periplasm and obtained with a vector based on a constitutive lambda-PL promoter. This antagonist can be useful for studies aiming at investigating the effects of a simultaneous inhibition of GH and prolactin signaling, as a potential anti-tumoral or anti-diabetic compound. G120R-hGH, synthesized using the W3110 E. coli strain, showed a yield of 1.34 ± 0.24 µg/ml/A600 (∼0.79 mg G120R-hGH/g of wet weight cells) after cultivation at 30 °C up to 3 A600 units and induction at 37 °C, for 6 h, with final 4.3 ± 0.3 A600. A laboratory scale purification was carried out using three chromatographic steps with a total yield of 32%, reaching 98% purity. The obtained protein was characterized by SDS-PAGE, Western Blotting, Mass spectrometry, RP-HPLC, HPSEC and in vitro proliferation bioassay. The proliferation assay, based on Ba/F3-LLP cells, shows that G120R-hGH (100 ng/ml) significantly inhibited (64%) the proliferative action of hGH (1 ng/ml). This is the first time that G120R-hGH is synthesized in bacterial periplasmic space and therefore correctly folded, without the initial methionine. The reasons for a divergent efficacy for antagonizing hGH versus hPRL is currently unknown and deserves further investigation.


Subject(s)
Amino Acid Substitution , Escherichia coli/metabolism , Human Growth Hormone , Membrane Proteins/antagonists & inhibitors , Periplasm/metabolism , Animals , Cell Line , Escherichia coli/chemistry , Escherichia coli/genetics , Human Growth Hormone/biosynthesis , Human Growth Hormone/chemistry , Human Growth Hormone/genetics , Human Growth Hormone/isolation & purification , Humans , Mice , Mutation, Missense , Periplasm/chemistry , Periplasm/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
12.
Methods Mol Biol ; 1466: 93-105, 2016.
Article in English | MEDLINE | ID: mdl-27473484

ABSTRACT

Purity determination of somatropin as a recombinant protein is important to ensure its safety and quality. This is carried out by capillary zone electrophoresis in double-injection mode using polybrene/chondroitin sulfate A double-coated capillaries. Modification of the capillary wall eliminates protein-wall interactions which results in improved accuracy and precision of the determinations. In the double-injection mode two somatropin samples are analyzed within a single electrophoretic run. Prior to the second injection, the first injected plug is electrophoresed for a predetermined time period in order to adjust the inter-plug distance. Here, the principle for the separation of somatropin charge variants is described.


Subject(s)
Electrophoresis, Capillary/methods , Human Growth Hormone/isolation & purification , Chondroitin Sulfates/chemistry , Hexadimethrine Bromide/chemistry , Human Growth Hormone/chemistry , Humans , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
13.
Bioconjug Chem ; 27(5): 1341-7, 2016 05 18.
Article in English | MEDLINE | ID: mdl-27108993

ABSTRACT

Human growth hormone (hGH) plays an important role during human development and is also an approved therapeutic for the treatment of several diseases. However, one major drawback of hGH is its short circulating half-life requiring frequent administration, which is inconvenient and painful for the patients. Recent publications indicate that circularization greatly increases the stability of proteins due to their protection from exoproteolytic attack and a higher thermal stability of the circular form. Using sortase A, a transpeptidase isolated from Staphylococcus aureus, we developed a single step solid-phase circularization and purification procedure resulting in a circular version of hGH with improved properties. We could show that circular hGH binds to the recombinant hGH receptor with binding kinetics similar to those of linear hGH and that circularization does not alter the biological activity of hGH in vitro. Besides, circular hGH showed almost complete resistance toward exoproteolytic attack and slightly increased thermal stability which could possibly translate into an extended plasma half-life. The single step solid-phase circularization and purification procedure is in principle a generic process, which could also be applied for other proteins that meet the requirements for circularization.


Subject(s)
Human Growth Hormone/chemistry , Human Growth Hormone/isolation & purification , Amino Acid Sequence , Aminoacyltransferases/metabolism , Bacterial Proteins/metabolism , Cysteine Endopeptidases/metabolism , Humans , Kinetics , Models, Molecular , Protein Stability , Protein Structure, Secondary , Staphylococcus aureus/enzymology
14.
Int J Biol Macromol ; 80: 400-9, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26116386

ABSTRACT

In this study, site-specific PEGylated human growth hormone (hGH) was prepared by microbial transglutaminase, modeled and characterized. To this end, the effects of different reaction parameters including reaction media, PEG:protein ratios, reaction time and pH value were investigated. PEG-hGH was purified by size exclusion chromatography method and analyzed by SDS-PAGE, BCA, peptide mapping, ESI and MALDI-TOF-TOF mass spectroscopy methods. Biophysical and biological properties of PEG-hGH were evaluated. Molecular simulation was utilized to provide molecular insight into the protein-receptor interaction. The optimum conditions that were obtained for PEGylation were phosphate buffer with pH of 7.4, 48 h of stirring and PEG:protein ratio of 40:1. By this method, mono-PEG-hGH with high reaction yield was obtained and PEGylation site was at Gln-40 residue. The circular dichroism and fluorescence spectrum indicated that PEGylation did not change the secondary structure while tertiary structure was altered. Upon enzymatic PEGylation, agonistic activity of hGH was preserved; however, Somavert(®), which is prepared by chemical PEGylation, is an antagonist form of protein. These data were confirmed by the total energy of affinity obtained by computational protein-receptor interaction. In conclusion, PEGylation of hGH was led to prepare a novel form of hormone with an agonist activity which merits further investigations.


Subject(s)
Human Growth Hormone/analogs & derivatives , Human Growth Hormone/chemical synthesis , Amino Acid Sequence , Bacterial Proteins/chemistry , Biocatalysis , Human Growth Hormone/isolation & purification , Humans , Molecular Docking Simulation , Molecular Sequence Data , Peptide Mapping , Polyethylene Glycols/chemistry , Transglutaminases/chemistry
15.
J Mol Recognit ; 28(7): 401-12, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25727088

ABSTRACT

The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes.


Subject(s)
Chromatography, Affinity/methods , Expressed Sequence Tags/chemistry , Recombinant Proteins/isolation & purification , Aminopeptidases/chemistry , Animals , Escherichia coli/genetics , Genetic Vectors , Glutathione Transferase/chemistry , Glutathione Transferase/genetics , Glutathione Transferase/isolation & purification , Heterocyclic Compounds/chemistry , Human Growth Hormone/chemistry , Human Growth Hormone/genetics , Human Growth Hormone/isolation & purification , Humans , Ions/chemistry , Metals/chemistry , Protein Structure, Tertiary , Proteolysis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Schistosoma japonicum , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
16.
Mol Biotechnol ; 57(3): 241-50, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25380986

ABSTRACT

The aim of this study was to achieve high-level production of the human growth hormone (hGH) in the prokaryotic expression system. In this regard, we performed cloning, expression, and purification of a synthetic hGH gene in BL21 (DE3) strain of E. coli. The hGH production was determined by SDS-PAGE and western blotting techniques, and then the protein concentration was determined by the Bradford assay. To gain insight into the effect of different nutrients on the growth of E. coli and hGH production, in a preliminary assessment nine different types of the basal medium were analyzed. The highest growth of E. coli and hGH production were observed in TB and SOB media. Accordingly, design of experiments was employed for screening the most significant nutrients, and central composite face design was applied for the optimization. The optimum medium consisted of yeast extract (10 g/L), tryptone (10 g/L), and K2HPO4 (2 g/L). The optimum hGH concentration was 391 mg/L, which was 3-fold higher than the hGH concentration in the LB basal medium (119 mg/L). This production rate is the highest hGH concentration reported in the IPTG-inducible expression systems.


Subject(s)
Escherichia coli/growth & development , Human Growth Hormone/biosynthesis , Human Growth Hormone/isolation & purification , Cloning, Molecular/methods , Culture Media/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Human Growth Hormone/genetics , Humans , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics
17.
PLoS One ; 9(12): e114351, 2014.
Article in English | MEDLINE | ID: mdl-25469649

ABSTRACT

Human growth hormone (hGH) is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the ß-trefoil superfamily, with ß domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people's interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs.


Subject(s)
Bombyx/chemistry , Human Growth Hormone/chemistry , Insect Proteins/chemistry , Amino Acid Sequence , Animals , Chromatography, Affinity , Human Growth Hormone/isolation & purification , Human Growth Hormone/physiology , Humans , Insect Proteins/isolation & purification , Insect Proteins/physiology , K562 Cells , Models, Molecular , Molecular Sequence Data , Pupa/chemistry , Structural Homology, Protein
18.
Protein Expr Purif ; 100: 40-7, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24859479

ABSTRACT

Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1 g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation, was confirmed by employing a Nb2 cell line proliferation assay.


Subject(s)
Cloning, Molecular/methods , Escherichia coli/genetics , Human Growth Hormone/genetics , Human Growth Hormone/isolation & purification , Cell Line , Cell Proliferation/drug effects , Chromatography, Affinity , Chromatography, Gel , Chromatography, Ion Exchange , Human Growth Hormone/chemistry , Human Growth Hormone/pharmacology , Humans , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Solubility , Thioredoxins/genetics , Thioredoxins/isolation & purification , Transformation, Bacterial
19.
Biotechnol Prog ; 30(5): 1057-64, 2014.
Article in English | MEDLINE | ID: mdl-24799458

ABSTRACT

This study demonstrates how the multimodal Capto adhere resin can be used in concert with calcium chloride or arginine hydrochloride as mobile phase modifiers to create a highly selective purification process for a modified human growth hormone. Importantly, these processes are shown to result in significant clearance of product related aggregates and host cell proteins. Furthermore, the steric mass action model is shown to be capable of accurately describing the chromatographic process and the aggregate removal. Finally, justification of the selected operating ranges is evaluated using the model together with Latin hypercube sampling. The results in this article establish the utility of multimodal chromatography when used with appropriate mobile phase modifiers for the downstream bioprocessing of a modified human growth hormone and offer new approaches for bioprocess verification.


Subject(s)
Chromatography, Liquid/methods , Human Growth Hormone/isolation & purification , Models, Chemical , Models, Statistical , Animals , Arginine , Calcium Chloride , Escherichia coli Proteins/chemistry , Human Growth Hormone/chemistry , Humans , Hydrophobic and Hydrophilic Interactions , Protein Aggregates , Rabbits , Static Electricity
20.
PLoS One ; 9(3): e89038, 2014.
Article in English | MEDLINE | ID: mdl-24614134

ABSTRACT

Human growth hormone (hGH) is synthesized by somatotroph cells of the anterior pituitary gland and induces cell proliferation and growth. This protein has been approved for the treatment of various conditions, including hGH deficiency, chronic renal failure, and Turner syndrome. Efficient production of hGH in Escherichia coli (E. coli) has proven difficult because the E. coli-expressed hormone tends to aggregate and form inclusion bodies, resulting in poor solubility. In this study, seven N-terminal fusion partners, hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), protein disulfide bond isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), were tested for soluble overexpression of codon-optimized hGH in E. coli. We found that MBP and hPDI tags significantly increased the solubility of the hormone. In addition, lowering the expression temperature to 18°C also dramatically increased the solubility of all the fusion proteins. We purified hGH from MBP-, PDIb'a'-, or Trx-tagged hGH expressed at 18°C in E. coli using simple chromatographic techniques and compared the final purity, yield, and activity of hGH to assess the impact of each partner protein. Purified hGH was highly pure on silver-stained gel and contained very low levels of endotoxin. On average, ∼37 mg, ∼12 mg, and ∼7 mg of hGH were obtained from 500 mL-cell cultures of Trx-hGH, MBP-hGH, and PDIb'a'-hGH, respectively. Subsequently, hGH was analyzed using mass spectroscopy to confirm the presence of two intra-molecular disulfide bonds. The bioactivity of purified hGHs was demonstrated using Nb2-11 cell.


Subject(s)
Human Growth Hormone/isolation & purification , Maltose-Binding Proteins/metabolism , Prokaryotic Cells/metabolism , Protein Disulfide-Isomerases/metabolism , Recombinant Fusion Proteins/isolation & purification , Thioredoxins/metabolism , Amino Acid Sequence , Animals , Cell Line , Cell Proliferation/drug effects , Escherichia coli/metabolism , Human Growth Hormone/chemistry , Human Growth Hormone/pharmacology , Humans , Molecular Sequence Data , Plasmids/metabolism , Prokaryotic Cells/drug effects , Protein Stability/drug effects , Protein Structure, Tertiary , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/pharmacology , Solubility , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Subcellular Fractions/drug effects , Subcellular Fractions/metabolism
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