Production Of Human Growth Hormone Research Articles

Comparison of Genomic and Episomal Expression of Recombinant Human Growth Hormone in Escherichia coli

Background: The production of recombinant human growth hormone is usually problematic in Escherichia coli, and achieving higher functional protein yields is economically important. Objectives: In this study, the chromosomal expression of recombinant human growth hormone (hGH) in the araBAD operon was investigated using Rosetta-gami and BL21 (DE3) competent cells under the regulation of arabinose and T7 promoters using arabinose inducer and IPTG, respectively. Subsequently, the expression of the plasmid-based protein was examined using pET28 (a) + plasmid in Rosetta-gami under the T7 promoter, and the results were compared. Methods: The lambda red technique was used to integrate the desired gene into the host genome. The Fh8 tag was used to increase the protein’s expression, solubility, and thermal resistance. Results: The recombinant BL21 (DE3) under the T7 promoter and the recombinant Rosetta-gami containing pET28-Fh8-hGH plasmid showed significant expression among the chromosome-based and plasmid-based strains, respectively. Preliminary studies on the solubility and thermal resistance of the produced proteins indicated the efficacy of the Fh8 tag in increasing the expression, solubility, and thermal resistance of the product. Conclusions: One advantage of the genomic expression approach is the stability of the gene in the genome. Also, the lack of the need to use antibiotics in production systems can effectively reduce the production costs of this widely used hormone. In addition, the presence of the Fh8 tag can facilitate and accelerate its purification process by increasing the heat resistance thermostability of growth hormone.

Development of a Bacterial FhuD-Lysozyme-SsrA Mediated Autolytic (FLSA) System for Effective Release of Intracellular Products

Developing effective bacterial autolytic systems for fast release of intracellular bioproducts could simplify purification procedures and help with the high throughput screening of mutant libraries in protein engineering. Here, we developed a fast and tightly regulated E.coli autolytic system, named the FhuD-lysozyme-SsrA mediated autolytic (FLSA) system, by integrating the secretion signal peptide, T7 lysozyme, and E.coli ClpX/P-SsrA protein degradation machinery. To decrease the cytotoxicity of leaky T7 lysozymes, the SsrA tag was fused to the C-terminus of T7 lysozyme to confer a tight regulation of its production. Using sfGFP as a reporter, we demonstrated that anchoring the Sec-Tat dual pathway signal peptide FhuD to the N-terminus of T7 lysozyme-SsrA could give the highest cell lysing efficiency. The optimization of the FLSA system indicated that weak alkaline conditions (pH 8.0) and 0.5% Triton X-100 could further increase the lysing efficiency by about 24%. The FLSA system was validated by efficient production of sfGFP and human growth hormone 1 (hGH1) in a shake flask, with a cell lytic efficiency of approximately 82% and 80%, respectively. Besides, the FLSA system was applied for large-scale fermentation, in which approximately 90% sGFP was released with a cell density OD600 of 110. Moreover, the FLSA system was also tested for α-amylase mutant library screening in microplates, and the results showed that intracellular α-amylase can be efficiently released out of cells for activity quantitation. In all, the FLSA system can facilitate the release of intracellular recombinant proteins into the cell culture medium, which has the potential to serve as an integrated system for large-scale production of recombinant targets and high throughput enzyme engineering in synthetic biology.

PMON68 Effect of an Amino-Acid Based Blend on Human Growth Hormone (hGH) and Fibromyalgia (FM) Symptoms

Abstract Fibromyalgia is a chronic pain sensitivity disorder that affects approximately 3-8% of the general population with symptoms that include fatigue and widespread musculoskeletal pain. Psychological and physical stressors are frequent drivers of fibromyalgia, and additional lockdown-associated stressors may worsen symptoms. Reduced production of human growth hormone (hGH) is evident in approximately 30% of individuals with fibromyalgia, which is hypothesized to contribute to common fibromyalgia symptoms. An orally administered amino acid supplement has been previously shown to improve endogenous hGH status in adults. We investigated if daily administration of the supplement would increase levels of endogenous insulin-like growth factor 1 (IGF-1), a surrogate marker of the body's hGH levels, and improve symptoms related to low-normal hGH production in a cohort of 84 individuals being treated for fibromyalgia. This is the full-cohort analysis of an open-label, single-arm study that investigated the effects of 24 weeks of daily oral administration of the supplement in individuals with low-normal hGH production. The primary endpoint was the change from baseline to endpoint (Week 24) in serum IGF-1. Additional endpoints included the change in body weight, clinical symptoms (assessed with the Revised Fibromyalgia Impact Questionnaire [FIQR], range 0-100, and Perceived Stress Scale [PSS], range 0-40), fasting cardiometabolic markers, tolerability, and safety. Participants continued to receive standard care. The study enrolled 84 fibromyalgia patients with low-normal age-adjusted IGF-1 serum levels. High mean±SD baseline FIQR and PSS scores of 76±16 and 32±5, respectively, indicated poor to moderate symptom management with standard care. All individuals completed 24 weeks of treatment. Serum IGF-1 levels increased with a Week 24 mean±SE change of 28.4±3.0 ng/mL (paired t-test p<0.001). Body weight was reduced with a Week 24 mean±SE change of -5.5±0.3 kg (p<0.001). The change from baseline in FIQR and PSS scores were -29.1±1.1 and -20.0±0.8, respectively (both p<0.001), indicating a substantial improvement. Statistically significant improvements from baseline were also observed for systolic and diastolic blood pressure, HbA1c, LDL and HDL cholesterol, and triglycerides (all p<0.001). The supplement was well tolerated; no adverse events were reported. Sustained augmentation of IGF-1 with the supplement may represent a unique method of improving clinical symptoms of fibromyalgia in individuals with low-normal hGH production. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Mammalian sialyltransferases allow efficient Escherichia coli-based production of mucin-type O-glycoproteins but can also transfer Kdo.

The prospect of producing human-like glycoproteins in bacteria is becoming attractive as an alternative to already-established but costly mammalian cell expression systems. We previously described an Escherichia coli expression platform that uses a dual-plasmid approach to produce simple mucin type O-glycoproteins: one plasmid encoding the target protein and another O-glycosylation machinery. Here, we expand the capabilities of our platform to carry out sialylation and demonstrate the high-yielding production of human interferon α2b and human growth hormone bearing mono- and disialylated T-antigen glycans. This is achieved through engineering an E. coli strain to produce CMP-Neu5Ac and introducing various α-2,3- and α-2,6 mammalian or bacterial sialyltransferases into our O-glycosylation operons. We further demonstrate that mammalian sialyltransferases, including porcine ST3Gal1, human ST6GalNAc2 and human ST6GalNAc4, are very effective in vivo and outperform some of the bacterial sialyltransferases tested, including Campylobacter jejuni Cst-I and Cst-II. In the process, we came upon a way of modifying T-Antigen with Kdo, using a previously uncharacterised Kdo-transferase activity of porcine ST3Gal1. Ultimately, the heterologous expression of mammalian sialyltransferases in E. coli shows promise for the further development of bacterial systems in therapeutic glycoprotein production.

Sleep deprivation and diet affect human GH gene expression in transgenic mice in vivo.

Human (h) growth hormone (GH) production studies are largely limited to effects on secretion. How pituitary hGH gene (hGH-N/GH1) expression is regulated is important in our understanding of the role hGH plays in physiology and disease. Here we assess for the first time the effect of sleep deprivation (SD) and high-fat diet (HFD) on hGH-N expression in vivo using partially humanized 171hGH/CS transgenic (TG) mice, and attempted to elucidate a role for DNA methylation. Activation of hGH-N expression requires interactions between promoter and upstream locus control region (LCR) sequences including pituitary-specific hypersensitive site (HS) I/II. Both SD and diet affect hGH secretion, but the effect of SD on hGH-N expression is unknown. Mice fed a HFD or regular chow diet for 3 days underwent SD (or no SD) for 6 h at Zeitgeber time (ZT) 3. Serum and pituitaries were assessed over 24 h at 6-h intervals beginning at ZT 14. SD and HFD caused significant changes in serum corticosterone and insulin, as well as hGH and circadian clock-related gene RNA levels. No clear association between DNA methylation and the negative effects of SD or diet on hGH RNA levels was observed. However, a correlation with increased methylation at a CpG (cytosine paired with a guanine) in a putative E-box within the hGH LCR HS II was suggested in situ. Methylation at this site also increased BMAL1/CLOCK-related nuclear protein binding in vitro. These observations support an effect of SD on hGH synthesis at the level of gene expression.

Ethanol fed-batch bioreactor operation to enhance therapeutic protein production in Pichia pastoris under hybrid-architectured ADH2 promoter

ABSTRACT We presented ethanol fed-batch bioreactor operations (FBBOs) designed for enhanced recombinant human growth hormone (rhGH) production in Pichia pastoris constructed with novel hybrid-architectured ADH2 promoter, PADH2-Cat8-L2. The parameters in the fed-batch fermentation of ethanol were investigated, and the boundaries of the production domain in terms of the design parameters were determined. Two FBBO design methods were used, and a platform aiming to adjust cellular metabolism for the generation of constant specific growth rate (μ) and substrate concentration (CEtOH) was established. The highest overall productivity (rP,ov) was generated with model-based continuous feed-stream (CFS) design with μpreD = 0.035 h-1 up to tmax ≤ 9 h as rP,ov = 3.0 mg L-1 h-1 and CrhGH = 34 mg L-1. The ethanol-stat FBBO at CEtOH,set = 0.5 g L-1 allowed increased product titer with high productivity and the highest CrhGH =90.1 mg L-1 at tmax = 24 h. The effect of CEtOH on specific rates for cell reactions was represented by the Haldane model in which the cell generation and rhGH production were simulated with low mean relative errors of 6.5 and 16%, respectively. The results indicate the need for an upgraded CFS design model with kinetic expressions involving substrate inhibition. We conclude that a two-step hybrid fed-batch design strategy to be started (i) with the initial-period of the model-based FBBO, in turn, proceeded (ii) with the controlled feeding with ethanol-stat FBBO enables a gradual approach to the hypothetical performance of FBBOs.

Hybrid-architectured double-promoter expression systems enhance and upregulate-deregulated gene expressions in Pichia pastoris in methanol-free media.

Double-promoter expression system (DPES) design as de novo metabolic engineering strategy enables fine-tuned and enhanced gene expression. We constructed a collection of monodirectional hybrid-architectured DPESs with engineered promoter variants PADH2-Cat8-L2 and PmAOX1 and with the naturally occurring promoter PGAP to enhance and upregulate-deregulated gene expressions in Pichia pastoris in methanol-free media. Reporter red fluorescent protein (mApple) and enhanced green fluorescent protein (eGFP) were expressed under PADH2-Cat8-L2 and PmAOX1 or PGAP, respectively, enabling the determination of the transcription period and strength of each constituent in the DPESs. We determined fluorescent protein expressions in batch cultivations on 2% (v/v) ethanol, excess glucose, and excess glycerol, and compared them with single-promoter expression systems constructed with PADH2-Cat8-L2, PmAOX1, and PGAP. The transcription- and expression-upregulation power of bifunctional DPESs was higher than that of twin DPESs (two-copy expression systems). Our findings answer long-standing questions regarding the high- (or multi-) copy clone results in the literature. Our first conclusion is that increasing identical components in the DPES architectures linearly increases the concentrations of cis-acting DNA sites and increases the demand for key transcription factors (TFs) that perturb their good coupling of supply and demand. The next is that the synthesis of some amino acids may create bottleneck(s) as rate-limiting amino acid(s) in recombinant protein synthesis. With bifunctional DPESs, each constituent upregulated the transcription and increased the expression and reduced the demand for the same TF(s) in the generation of novel regulatory circuits, due to the increased number of nonidentical cis-acting DNA sites. We tested superior DPES performances in extracellular human growth hormone (rhGH) production. Thereby, the indications related to the rate-limiting amino acids were verified. Compared with its constituents PADH2-Cat8-L2 and PmAOX1, the bifunctional DPES4 enhanced rhGH production by 1.44- and 2.02-fold, respectively. The DPES design method, with its constraint and parameters, enables the generation of promising r-protein production platforms with high impact on industrial-scale production processes and opens up new avenues for research in yeasts. KEY POINTS: • Design method with the constraint and parameters for the construction of the DPESs is presented. • Hybrid-architectured de novo DPESs are designed to enhance and fine-tune gene expression. • Bifunctional DPESs demonstrate enhanced transcription and expression. • Twin DPESs linearly increase cis-acting DNA sites and consequently increase the demand for the same TFs. • Bifunctional DPESs enable good coupling of supply and demand to bind with TFs. • Ethanol-controlled Snf1 pathway and crosstalk enable fine-tuned transcription and enhanced expression.

Engineered small metal-binding protein tag improves the production of recombinant human growth hormone in the periplasm of Escherichiacoli.

Fusion proteins play an important role in the production of recombinant proteins in Escherichia coli. They are mostly used for cytoplasmic expression since they can be designed to increase the solubility of the target protein, which then can be easily purified via affinity chromatography. In contrast, fusion proteins are not usually included in construct designs for periplasmic production. Instead, a signal sequence is inserted for protein transport into the periplasm and a C‐terminal his‐tag added for subsequent purification. Our research group has proposed the small metal‐binding protein (SmbP) isolated from the periplasm of Nitrosomonas europaea as a new fusion protein to express recombinant proteins in the cytoplasm or periplasm of E. coli. SmbP also allows purification via immobilized metal affinity chromatography using Ni(II) ions. Recently, we have optimized the periplasmic production of proteins tagged with SmbP by exchanging its native signal peptide with one taken from pectate lyase B (PelB), substantially increasing the amount of protein produced. In this work, we have expressed and purified soluble bioactive human growth hormone (hGH) tagged with PelB‐SmbP and obtained the highest periplasmic production reported for this protein so far. Its activity, tested on Nb2‐11 cells, was equivalent to commercial growth hormone at 50 ng·mL−1. Therefore, we strongly recommend the use of PelB‐SmbP as a protein tag for the expression and purification of hGH or other possible target proteins in the periplasm of E. coli.

Escherichia coli Can Adapt Its Protein Translocation Machinery for Enhanced Periplasmic Recombinant Protein Production

Recently, we engineered a tunable rhamnose promoter-based setup for the production of recombinant proteins in E. coli. This setup enabled us to show that being able to precisely set the production rate of a secretory recombinant protein is critical to enhance protein production yields in the periplasm. It is assumed that precisely setting the production rate of a secretory recombinant protein is required to harmonize its production rate with the protein translocation capacity of the cell. Here, using proteome analysis we show that enhancing periplasmic production of human Growth Hormone (hGH) using the tunable rhamnose promoter-based setup is accompanied by increased accumulation levels of at least three key players in protein translocation; the peripheral motor of the Sec-translocon (SecA), leader peptidase (LepB), and the cytoplasmic membrane protein integrase/chaperone (YidC). Thus, enhancing periplasmic hGH production leads to increased Sec-translocon capacity, increased capacity to cleave signal peptides from secretory proteins and an increased capacity of an alternative membrane protein biogenesis pathway, which frees up Sec-translocon capacity for protein secretion. When cells with enhanced periplasmic hGH production yields were harvested and subsequently cultured in the absence of inducer, SecA, LepB, and YidC levels went down again. This indicates that when using the tunable rhamnose-promoter system to enhance the production of a protein in the periplasm, E. coli can adapt its protein translocation machinery for enhanced recombinant protein production in the periplasm.

Improvement in the downstream processing of rhGH production in Pichia pastoris

Production of human proteins in Pichia pastoris has significant advantages. However, there is still need for improvement in various stages of its downstream processing like clarification and purification. In fact downstream processes are usually the most critical part of production of biotech products. This work aimed to evaluate the effect of two steps added to the downstream processes of human growth hormone (hGH) production in Pichia pastoris. Firstly the effect of clarification, with activated carbon, on capture of hGH by ion exchange chromatography (IEC) was investigated. For this purpose, a clarification process using activated carbon was used to remove process contaminants like pigments. The clarified sample was applied to the IEC column and the recovery of hGH, following IEC, was assessed using SDS-PAGE, Bradford protein assay and area under the curve (AUC). The obtained results showed that the AUC values were 2.81 and 5.61 for the with- and without-treatment samples, respectively. Protein recovery of clarified sample with activated carbon was 541 mg in comparison with 328 mg for the sample without treatment. The yield of IEC was also improved from 50.46% to 83.23% following treatment with activated carbon. Secondly, the effect of three concentrations of ammonium sulfate in the binding buffer on resolution of hGH upon elution on hydrophobic interaction chromatography (HIC) was investigated. Biological activity was used as the main criterion for evaluation of purified hGH using HIC. The obtained results indicated that by increasing the concentration of ammonium sulfate form 1 to 3 mol/L, resolution of hGH was improved, as the purified fraction using 3 mol/L of ammonium sulfate showed a specific activity of 3.1IU/mg. So the results of the present study demonstrated that activated carbon is a promising candidate for efficient clarification of recombinant hGH and improving the efficiency of the capture step. Therefore, it can be considered by biotech companies as a cost-effective and sustainable clarification procedure of recombinant proteins from high cell density cultures. This study also revealed that 3% ammonium sulfate has a positive effect on the separation of hGH variants with the desired biological activity.

Hybrid fed‐batch bioreactor operation design: control of substrate uptake enhances recombinant protein production in high‐cell‐density fermentations

AbstractBACKGROUNDThe hybrid fed‐batch bioreactor (HFBB) operation is a novel design method for high‐cell‐density fermentations. It employs fed‐batch and batch operations, starting with fed‐batch that in turn shifts to the next, and proceeds in successive cycles to enhance production and productivity by controlling the substrate uptake rate.RESULTSThe method was tested for the production of recombinant human growth hormone (rhGH) in Pichia pastoris under GAP promoter with carbon sources of (i) glucose and (ii) pre‐treated hydrolyzed sugar‐beet molasses containing equimolar glucose and fructose. The superior strategies were: (i) GH2, a HFBB with glucose feeding composed of ΔtFB = 1.5 h fed‐batch periods with continuous‐feed stream designed with the pre‐determined growth rate µ = 0.10 h−1, which shift in turn to ΔtB = 0.5 h batch periods; and (ii) MH2, a HFBB with molasses feeding designed in a similar way to GH2. The highest rhGH production attained in GH2 was 611 mg dm−3 at 8 h, whereas it was 625 mg dm−3 at 13.5 h in MH2. In GH2, rhGH production increased 1.37‐fold in half the cultivation time of GH2. However, in MH2 with dual carbon sources enriched with other substrates, higher rhGH production was obtained at the cost of lower productivity.CONCLUSIONThe HFBB is a powerful design method with a single carbon source which improves the productivity of high‐cell‐density fermentations. However, with molasses upon shifting to batch operations, the uptake of accumulated fructose is continued. Thus, the glamour of the HFBB is hindered for complex media fermentations to an extent, because multi‐carbon sources contribute to the growth during the batch periods by lengthening the cultivation time. © 2018 Society of Chemical Industry

Influence of autocrine growth hormone on NF-κB activation leading to epithelial-mesenchymal transition of mammary carcinoma.

Progression of breast cancers often depends on hormones among which human growth hormone is prominently involved in breast cancer progression. Earlier studies have reported constitutive activation of nuclear factor-κB, a key regulator of growth hormone receptor-mediated signaling pathway in breast carcinoma, but the precise molecular mechanisms are still elusive. In this study, we investigated the effect of human growth hormone on nuclear factor-κB activation and epithelial-mesenchymal transition in breast carcinoma. Our results explored that autocrine production of human growth hormone enhances cellular proliferation by the activation of nuclear factor-κB (65 kDa) and downregulation of E-cadherin expression. Furthermore, enhanced nuclear factor-κB expression significantly increases cell proliferation and diminishes apoptosis in MCF-7 cell line. Increased expression of nuclear factor-κB significantly enhances mammary carcinoma cell migration and invasion stimulated by autocrine human growth hormone, which results in epithelial-mesenchymal transition of MCF-7 cells. In conclusion, our study revealed the influence of human growth hormone on nuclear factor-κB activity and epithelial-mesenchymal transition in mammary carcinoma. Our findings will help to understand molecular role of "growth hormone-nuclear factor-κB axis" in mammary carcinogenesis which may facilitate the discovery of suitable pathway inhibitors for disease treatment.

Do anabolic nutritional supplements stimulate human growth hormone secretion in elderly women with heart failure?

Growth hormone treatment has gained attention over the past decade as a treatment for heart failure. Human growth hormone (HGH) must be administered by injections (usually daily), so there is considerable advantage to stimulation of endogenous secretion by amino acid‐based nutritional supplementation. However, studies investigating the effect of amino acid (AA) supplementation show conflicting results. Therefore, in this study we aimed to investigate the effect of nutritional supplementation on HGH production in elderly women with heart failure. Eight elderly women with heart failure participated in this randomized cross‐over study. Plasma HGH concentration was measured before and for 4 h following ingestion of a mixture of protein, carbohydrate, and fat or an AA beverage. HGH concentration was determined with ELISA kits and AA concentrations were analyzed by Liquid Chromatography–Mass Spectrometry (LCMS). Linear mixed models was performed to analyze the effect of time, treatment, and interaction. Plasma arginine and lysine concentrations were significantly higher after consumption of the AA drink compared to the mixture of protein, carbohydrate, and fat. Nonetheless, only ingestion of the protein, carbohydrate, and fat mixture (meal replacement) increased HGH concentration. HGH concentration was increased in elderly women with heart failure following consumption of a meal replacement containing protein, carbohydrate, and fat. Consumption of a mixture of amino acids failed to increase HGH concentration despite significantly greater elevations in plasma amino acid concentrations, including arginine and lysine. The stimulatory effect of the protein/carbohydrate/fat mixture was presumably mediated by factors other than increases in free amino acid concentrations.

Analyses of extracellular protein production in Bacillus subtilis – II: Responses of reaction network to oxygen transfer at transcriptional level

Oxygen transfer influences intracellular fluxes which are orchestrated by genome and its transcription in Bacillus subtilis throughout fermentation in recombinant human growth hormone (rhGH) production. Responses of B. subtilis reaction network to oxygen transfer were analysed at transcriptional level with determined transcriptome and calculated intracellular fluxes by the reconstructed genome scale model iBsu1144rhGH based on updated gene-enzyme-reaction data. iBsu1144rhGH employing 1067 reactions linked to 719 genes was solved using time-profiles of rhGH fermentation data at three oxygen transfer conditions from low- to high- to create perturbations on the intracellular reaction-network. Insights obtained from transcriptional and metabolic responses were used to determine metabolic engineering sites. Flux variability analyses results reveal that 305 reactions are active, linked to 346 genes so called substantial genes; and, 730 reactions linked to 536 genes blocked in rhGH synthesis were determined with their enzymes, genes, and reaction fluxes to create a platform for guided gene deletions. Effects of oxygen transfer on the transcriptome of r-B. subtilis carrying hGH gene were investigated further with one-way analysis of variance with the confidence greater than 99.5% and the expression ratios greater than 3. Fifty probe sets were found to be significantly affected from oxygen transfer where eight genes performed different transcriptional and metabolic responses to oxygen transfer and determined as metabolic engineering sites besides scoC and degU.

Construction of the plasmid-free strain for human growth hormone production.

The E.coli strain, overproducing human growth hormone (hGH) was made by integration of the hGH gene under the control of T7 promoter into the chromosomal LacZ gene of BL21(DE3) via lambda Red recombineering. The strain gave higher productivity (50mg·L(-1)·OD550(-1)) and better growth characteristics than the corresponding strain in which the same hGH expression cassette was placed in a plasmid. The protein produced by the plasmid-free strain was purified and characterized to be hGH. The results demonstrates that a plasmid-free recombinant strain having a single-copy gene expression cassette in the chromosome could provide better gene activity regulation, higher productivity, superior growth characteristics, as well as more stringent control of the gene sequence invariance than a plasmid-based strain.

Feeding strategy design for recombinant human growth hormone production by Bacillus subtilis.

Defined and semi-defined medium-based feeding strategies were developed to enhance recombinant human growth hormone (rhGH) production by Bacillus subtilis BGSC-1A178 (scoC (-)) strain carrying pMK4::pre(subC)::hGH. Defined medium-based feeding strategies were designed by exponential feeding of glucose and (NH4)2HPO4 at two pre-determined specific growth rates, µ 0=0.10 and 0.17h(-1). Semi-defined medium-based feeding strategies were designed by exponential feeding of substrate solution consisting of glucose, (NH4)2HPO4, peptone, and tracesalt solution (PTM1) at three pre-determined specific growth rates, µ 0=0.10, 0.17, and 0.25h(-1). At all the strategies applied, transition cultivation time from batch to fed-batch operation was t T=4h. The highest rhGH concentration was obtained as C rhGH=0.5gL(-1) with semi-defined medium-based feeding strategy designed with µ 0=0.25h(-1) using feed substrate stock solution containing 200gL(-1) glucose, 117gL(-1) (NH4)2HPO4, 100gL(-1) peptone, and 5mLL(-1) PTM1 at t=22h when the cell concentration reached to C X=8.29g L(-1). The overall product and cell yields on glucose were obtained as [Formula: see text]=7.21mgg(-1) and [Formula: see text]=0.12gg(-1), respectively. The results indicate the requirement of designing continuous feed stream in fed-batch production to enhance rhGH production by r-B. subtilis.

Methanol feeding strategy design enhances recombinant human growth hormone production by Pichia pastoris

ABSTRACTBACKGROUNDThe effects of designed methanol and co‐substrate feeding strategies on fed‐batch recombinant human growth hormone (rhGH) production by Pichia pastoris hGH‐Mut+ were investigated.RESULTSThe highest cell concentration was obtained as 157 g L−1 with the exponential feeding strategy where methanol was fed at a pre‐calculated feeding rate based on pre‐determined µ0 = 0.03 h−1, and mannitol concentration was kept constant at 50 g L−1 for t=0–6 h. The highest rhGH concentration was obtained as 1.3 g L−1 at t=54 h with the methanol‐stat feeding strategy, where no co‐substrate was used and the methanol concentration was kept at 5 g L−1 in the medium during the production phase. Thereafter, in another methanol‐stat feeding strategy with pulse‐fed sorbitol, the highest rhGH concentration was obtained as 1.2 g L−1 at t=42 h.CONCLUSIONThe methanol‐stat fed‐batch method keeping methanol concentration constant at 5 g L−1 in the production medium increased rhGH production at least 2‐fold compared with that of the exponential feeding strategies. In the designed methanol‐stat feeding strategies, use of sorbitol as co‐substrate decreased methanol demand of the cells 2‐fold, increased the overall product yield on the substrate 1.6‐fold, shortened the cultivation time by 12 h and increased the productivity. Thus, methanol‐stat feeding with co‐substrate sorbitol was found to be a favorable fed‐batch strategy for rhGH production by Pichia pastoris under alcohol oxidase I promoter. © 2014 Society of Chemical Industry

In Silico Evaluation of Different Signal Peptides for the Secretory Production of Human Growth Hormone in E. coli

Various advantages of protein secretion have prompted scientists to search for secretory production of heterologous proteins. Signal peptides are one of the most important factors for prosperous secretion of the recombinant proteins. The aim of this study was to evaluate 23 different signal peptides and theoretically determine suitable ones for secretory production of the human growth hormone (hGH) in the E. coli host. The signal peptide sequences and the precise location of their cleavage sites were predicted using SignalP 4.0 server. Accordingly, six of the signal peptides, including hGH, major outer membrane lipoprotein, protease VII, protein TolB, periplasmic protein TorT and beta-lactamase TEM were excluded from the further study, due to their inappropriate cleavage scores. Different physico-chemical properties, which are essential for selecting a proper signal peptide, were evaluated using ProtParam and Solpro as the most accurate and reliable servers. Computational analysis of the above-mentioned factors, indicates that outer membrane protein C, fimbrial chaperone SfmC, outer membrane protein F and disulfide interchange protein DsbA can theoretically be suitable signal peptides for hGH secretion.

Production of recombinant human growth hormone conjugated with a transcytotic peptide in Pichia pastoris for effective oral protein delivery.

Among the possible delivery routes, the oral administration of a protein is simple and achieves high patient compliance without pain. However, the low bioavailability of a protein drug in the intestine due to the physical barriers of the intestinal epithelia is the most critical problem that needs to be solved. To overcome the low bioavailability of a protein drug in the intestine, we aimed to construct a recombinant Pichia pastoris expressing a human growth hormone (hGH) fusion protein conjugated with a transcytotic peptide (TP) that was screened through peroral phage display to target goblet cells in the intestinal epithelia. The TP-conjugated hGH was successfully produced in P. pastoris in a secreted form at concentrations of up to 0.79 g/l. The function of the TP-conjugated hGH was validated by in vitro and in vivo assays. The transcytotic function of the TP through the intestinal epithelia was verified only in the C terminus conjugated hGH, which demonstrated the induction of IGF-1 in a HepG2 cell culture assay, a higher translocation of recombinant hGH into the ileal villi after oral administration in rats and both IGF-1 induction and higher body weight gain in rats after oral administration. The present study introduces the possibility for the development of an effective oral protein delivery system in the pharmaceutical and animal industries through the introduction of an effective TP into hGH.

Production of recombinant human growth hormone by rCHO cells in a depth filter perfusion system

Human growth hormone is a single-chain polypeptide produced commercially from recombinant animal cells as well as recombinant microorganisms. Its increased applications have requested development of highly efficient production systems using particularly animal cells. Depth filter perfusion system (DFPS) has been developed and successfully used for production of recombinant proteins such as antibodies from recombinant Chinese hamster ovary (rCHO) cells. In this study, rCHO cells expressing recombinant human growth hormone (rhGH) were successfully cultivated in the DFPS for 2,200 h. Parameters affecting the performance of the DFPS for rhGH production were investgated. The depth filter with 40 µm pores was selected for stable operation. The shifts of culture temperature and pH were tested at 37 and 33℃, and 7.2 and 7.0, respectively. In the begining of the culture, more than 80% of the seeded cells were immobilized in 200 min by medium/cell circulation. When the culture temperature was lowered from 37 to 33℃, about 50% increase of the volumetric productivity (VP) at a perfusion rate of 2.0/day was achieved, and the VP reached 168 mg/L/d at perfusion rate of 3.0/day, showing the benefit of cultures in low temperature. In contrast, when pH was shifted from 7.2 to 7.0, maintaining rhGH concentration at about 60 mg/L and decreased perfusion rate from 2.0 to 1.0/day, the VP dropped to 50%. As the DFPS showed stable and efficient production of rhGH for long-term periods from this study, it can be an attractive choice for production of rhGH from recombinant animal cells.