Expression of functional recombinant human growth hormone in transgenic soybean seeds

We produced human growth hormone (hGH), a protein that stimulates growth and cell reproduction, in genetically engineered soybean [Glycine max (L.) Merrill] seeds. Utilising the alpha prime (α') subunit of β-conglycinin tissue-specific promoter from soybean and the α-Coixin signal peptide from Coix lacryma-jobi, we obtained transgenic soybean lines that expressed the mature form of hGH in their seeds. Expression levels of bioactive hGH up to 2.9% of the total soluble seed protein content (corresponding to approximately 9?g?kg(-1)) were measured in mature dry soybean seeds. The results of ultrastructural immunocytochemistry assays indicated that the recombinant hGH in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Specific bioassays demonstrated that the hGH expressed in the soybean seeds was fully active. The recombinant hGH protein sequence was confirmed by mass spectrometry characterisation. These results demonstrate that the utilisation of tissue-specific regulatory sequences is an attractive and viable option for achieving high-yield production of recombinant proteins in stable transgenic soybean seeds.     

Plant‐produced human growth hormone shows biological activity in a rat model

Plants have been shown to be efficient systems for expressing a wide range of recombinant proteins from various origins. Here, using a plant virus‐based expression vector to produce human growth hormone (hGH) in Nicotiana benthamiana plants, we demonstrate, for the first time, that the plant‐produced hGH (pphGH) is biologically active in a hypophysectomized rat model. We observed an average weight gain of ～17 g per animal in a group of 10 animals that were injected subcutaneously with pphGH with 60 μg/dose for 10 days. With the increasing demand for hGH, accompanied with the need to make this recombinant protein available to a wider population at a more reasonable cost, plants provide a feasible alternative to current production platforms. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Expression of human growth hormone in transgenic rice cell suspension culture

Human growth hormone (hGH), a pituitary-derived polypeptide, evidences a wide range of biological functions, including protein synthesis, cell proliferation, and metabolism. A synthetic hGH gene (shGH) has been synthesized on the basis of plant-optimized codon usage via an overlap PCR strategy and located in a plant expression vector under the control of the rice amylase 3D (Ramy3D) promoter, which is induced by sugar starvation. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Donjin) via particle bombardment transformation methods. The integration of the shGH gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification and shGH expression in transgenic rice suspension cells was confirmed via Northern blot analysis. The shGH protein was detected in the transgenic rice cell suspension culture medium following induction with sugar starvation, using Western blot analysis. The quantity of shGH that accumulated in the transgenic rice cell suspension medium was 57 mg/l. The shGH accumulated in the transgenic rice cell suspension culture medium evidenced a biological activity similar to that of Escherichia coli-derived recombinant hGH. These results indicate that the shGH was generated and accumulated in the transgenic rice cell suspension culture medium, and manifested biological activity.     

Protein production from recombinant protein bodies

In this study, we describe a process for protein expression and purification from plants and insect cells based on the accumulation of recombinant proteins in protein bodies. This technology is using Zera^®, which sequence has the capacity to trigger in vivo the formation of dense, non-secretory storage protein body-like organelles derived from the endoplasmic reticulum (ER). With this method, recombinant human growth hormone (hGH) was expressed and purified from protein bodies accumulated in plants (Nicotiana benthamiana) and in insect cells (Spodoptera frugiperda). We found that recombinant Zera-hGH are stored in large quantity inside those proteins bodies and can be easily recovered during a one-step process from plant and insect cell biomass. After solubilization of recombinant protein bodies and cleavage of Zera tag from the fusion protein, active hGH was finally purified by a single chromatography step. These results indicate that recombinant proteins derived from Zera-fusion could provide both an efficient protein production system and eased purification downstream process.     

Expression of a recombinant human erythropoietin in suspension cell cultures of Arabidopsis, tobacco and Medicago

In the last two decades, the use of plants to produce recombinant proteins and particularly biopharmaceuticals has become an attractive alternative to established systems. This is due to advantages in scalability, economy and safety. In addition the expression of recombinant proteins in plants can also be achieved utilizing in vitro cell suspensions with all the advantages such systems confer, such as product consistency, production ??on demand?? and the ability to perform the entire process according to good manufacturing practices. In this study we have produced the glycosylated human hormone Erythropoietin (EPO), in Medicago truncatula and Arabidopsis thaliana plants and also in cultured cell lines of tobacco, Medicago and Arabidopsis. We have also tested two different versions of the protein, one with a KDEL tag for targeted expression in the Endoplasmic Reticulum, and an untagged version expected to be secreted to the apoplast. The recombinant protein was detected in the plant leaf extracts and in the cultured cell lines. In the latter, the rEPO was detected in the cell extracts and in the spent culture medium. It was possible to recover the KDEL version of rEPO from crude cell extracts by nickel affinity chromatography, however the secreted form did not bind to the Ni- agarose beads which may indicate a possible internalization of the his-tag in the folded protein. Although the yield of rEPO obtained in cell suspensions was not as high as expected, the protein was successfully produced and secreted into the culture medium, reinforcing that plant cell suspension cultures are a promising system for production of human biopharmaceuticals.     

Effect of shear stress on expression of a recombinant protein by Chinese hamster ovary cells

A flow chamber was used to impart a steady laminar shear stress on a recombinant Chinese hamster ovary (CHO) cell line expressing human growth hormone (hGH). The cells were subjected to shear stress ranging from 0.005 to 0.80 N m(-2). The effect of shear stress on the cell specific glucose uptake, cell specific hGH, and lactate productivity rates were calculated. No morphological changes to the cells were observed over the range of shear stresses examined. When the cells were subjected to 0.10 N m(-2) shear in protein-free media without Pluronic F-68, recombinant protein production ceased with no change in cell morphology, whereas control cultures were expressing hGH at 0.35 microg/10(6 )cells/h. Upon addition of the shear protectants, Pluronic F-68 (0.2% [w/v]) or fetal bovine serum (1.0% [v/v] FBS), the productivity of the cells was restored. The effect of increasing shear stress on the cells in protein-free medium containing Pluronic F-68 was also investigated. Cell specific metabolic rates were calculated for cells under shear stress and for no-shear control cultures performed in parallel, with shear stress rates expressed as a percentage of those obtained for control cultures. Upon increasing shear from 0.005 to 0.80 N m(-2), the cell specific hGH productivity decreased from 100% at 0.005 N m(-2) to 49% at 0.80 N m(-2) relative to the no-shear control. A concurrent increase in the glucose uptake rate from 115% at 0.01 N m(-2) to 142% at 0.80 N m(-2), and decreased lactate productivity from 92% to 50%, revealed a change in the yield of products from glucose compared with the static control. It was shown that shear stress, at sublytic levels in medium containing Pluronic F-68, could decrease hGH specific productivity.     

Extracellular production of an intact and biologically active human growth hormone by the Bacillus brevis system

The characteristic features of the Bacillus brevis system are very high productivity of heterologous proteins and very low extracellular protease activity. However, degradation of some heterologous proteins, especially mammalian proteins, can be observed and resulted in a lowering of protein productivity. By using a mutant expressing low levels of proteases and the addition of EDTA to the medium, intact human growth hormone (hGH) was successfully produced with the B. brevis system. Signal peptide modification with higher basicity in the amino terminal region and higher hydrophobicity in the middle region brought about a twelve-fold increase in hGH production. The hGH yield was further elevated to 240 mg L⁻¹ by optimization of culture conditions. Thus, biologically active and mature hGH can be efficiently produced directly in the medium with the B. brevis system. Received 06 March 1997/ Accepted in revised form 03 July 1997     

Biosynthesis and secretion of recombinant human growth hormone in Pichia pastoris

Mature human growth hormone (hGH) cDNA was cloned by homologous recombination into the yeast Pichia pastoris genome. The hGH gene expression was placed under the control of the methanol-inducible alcohol oxidase 1 (AOX1) gene promoter and the Saccharomyces cerevisiae -factor signal sequence to direct the secretion of recombinant human growth hormone (rhGH) into the growth medium. O₂-limited induction of recombinant yeast strains in shake tubes with 3 ml of culture medium produced up to 11 mg rhGH l^–1, while high cell density cultures using a 2-l bioreactor produced about 49 mg rhGH l^–1 achieving 40% of total protein of the culture medium supernatant.     

Enhanced accumulation of secreted human growth hormone by transgenic tobacco cells correlates with the introduction of an N-glycosylation site

Extracellular secretion of recombinant proteins from plant cell suspension culture will simplify the protein purification procedure and greatly reduce the production cost. Our early work indicated that presence of hydroxyproline-O-glycosylation at the C- or N-terminus of the target protein boosted the secreted yields in the culture medium. Inspired by early successes, we tested the possibility of introducing an N-glycosylation site to facilitate the secretion of human growth hormone (hGH) from cultured tobacco cells. Three N-glycosylated hGH fusion proteins, designated NAS-EK-hGH, NAS-Kex2-hGH and hGH-NAS, were expressed in tobacco BY-2 cells. Where NAS denotes the “Asn-Ala-Ser” consensus sequence for N-glycosylation; EK denotes an enterokinase cleavage site and Kex2 a sequence to be cleaved by a Golgi-localized Kex2p-like protease. Our results indicated that a single N-glycan attached either at the N-terminus or C-terminus of hGH correlated with enhanced extracellular accumulation of the transgenic proteins; the secreted yield of NAS-EK-hGH and hGH-NAS was 70-90 fold greater than the control targeted, non-glycosylated hGH. NAS-Kex2-hGH was subject to partial cleavage of the N-glycan tag at the Kex2 site in Golgi apparatus, and therefore gave lower yields than the other two constructs.     

Epi-CHO, an episomal expression system for recombinant protein production in CHO cells

This study describes the development of a transient expression system for CHO cells based on autonomous replication and retention of transfected plasmid DNA. A transient expression system that allows extrachromosomal amplification of plasmids permits more plasmid copies to persist in the transfected cell throughout the production phase leading to a significant increase in transgene expression. The expression system, named Epi-CHO comprises (1) a CHO-K1 cell line stably transfected with the Polyomavirus (Py) large T (LT) antigen gene (PyLT) and (2) a DNA expression vector, pPyEBV encoding the Py origin (PyOri) for autonomous plasmid amplification and encoding Epstein-Barr Virus (EBV) nuclear antigen-1 (EBNA-1) and OriP for plasmid retention. The CHO-K1 cell line expressing PyLT, named CHO-T was adapted to suspension growth in serum-free media to facilitate large-scale transient transfection and recombinant gene expression. Enhanced green fluorescent protein (EGFP) and human growth hormone (hGH) were used as reporter proteins to demonstrate transgene expression and productivity. Transfection of suspension-growing CHO-T cells with the vector pPyEBV encoding hGH resulted in a final concentration of 75 mg L(-1) of hGH in culture supernatants 11 days following transfection.     

Bioreactor strategies for improving production yield and functionality of a recombinant human protein in transgenic tobacco cell cultures

Plant cell culture production of recombinant products offers a number of advantages over traditional eukaryotic expression systems, particularly if the product can be targeted to and purified from the cell culture broth. However, one of the main obstacles is product degradation by proteases that are produced during cell culture, and/or the loss of biological activity of secreted (extracellular) products as a result of alteration in the protein conformation. Because proteolysis activity and target protein stability can be significantly influenced by culture conditions, it is important to evaluate bioprocess conditions that minimize these effects. In this study, a bioreactor strategy using a protocol involving pH adjustment and medium exchange during plant cell culture is proposed for improving the production of functional recombinant alpha(1)-antitrypsin (rAAT), a human blood protein, produced using several alternative expression systems, including a Cauliflower mosaic virus (CaMV) 35S constitutive promoter expression system, a chemically inducible, estrogen receptor-based promoter (XVE) expression system, and a novel Cucumber mosaic virus (CMV) inducible viral amplicon (CMViva) expression system developed by our group. We have demonstrated that higher medium pH help reduce protease activity derived from cell cultures and improve the inherent stability of human AAT protein as well. This strategy resulted in a fourfold increase in the productivity of extracellular functional rAAT (100 microg/L) and a twofold increase in the ratio of functional rAAT to total rAAT (48%) in transgenic N. benthamiana cell cultures using a chemically inducible viral amplicon expression system.     

Where, oh where has my protein gone?

A recent publication by R. Chikwamba and colleagues highlights interesting issues in recombinant protein expression in transgenic plants. In the study they expressed a bacterial antigen in maize seed and obtained aberrant localization data. This work is of great importance to the biotechnology industry and raises fascinating questions in plant cell biology that require creative thinking.     

Heat-Induced Production of Human Growth Hormone by High Cell Density Cultivation of Recombinant Escherichia Coli

The temperature-induced, over-expression of the human growth hormone gene in a recombinant E. coli during high cell density cultivation is reported. Human growth hormone (hGH) production and stability were tested under different heat shock conditions. Cell densities were 25 and 60 g l(-1) in a pH-stat fed-batch mode in defined and complex medium, respectively, and the fermentation time was decreased from 41 to 32 h. hGH was produced at 2 g l(-1) in complex medium. By using glycerol as main carbon source in the complex medium with exponential feeding, cell density and hGH production were increased to 100 g l(-1) and 2.7 g l(-1), respectively.     

Comparison of the Escherichia coli proteomes for recombinant human growth hormone producing and nonproducing fermentations

Two-dimensional electrophoretic analyses of Escherichia coli cells producing recombinant human growth hormone (Nutropin) in fermentations were conducted. The resulting two-dimensional protein profiles were compared with those of nonproducing (blank) cells. A qualitative comparison was performed to address regulatory issues in the biopharmaceutical industry, and a semiquantitative comparison was performed to reveal information about the physiological state of the cells. The protein spots unique to production fermentation profiles were all related to recombinant human growth hormone (hGH); these included intact hGH, charge variants of hGH, and a proteolytically cleaved form of hGH, as expected. There were no E. coli host cell proteins unique to either the production or blank fermentation profiles. Rather, all detectable differences in E. coli proteins were quantitative in nature. Specifically, the levels of IbpA (inclusion body binding protein A), Ivy (inhibitor of vertebrate lysozyme), and a cleaved form of GroEL (Hsp60 homolog) were higher in hGH production profiles, whereas the levels of GlmU protein and PspA (phage shock protein A) were higher in blank profiles. In general, the high degree of similarity between proteomes for hGH-producing and nonproducing cells suggests that E. coli proteins from a nonproducing (blank) fermentation are appropriate for eliciting antibodies that are then used in immunoassays to measure host cell proteins in samples from production fermentations.     

Production of human growth hormone in transgenic rice seeds: co-introduction of RNA interference cassette for suppressing the gene expression of endogenous storage proteins

Rice seeds are potentially useful hosts for the production of pharmaceutical proteins. However, low yields of recombinant proteins have been observed in many cases because recombinant proteins compete with endogenous storage proteins. Therefore, we attempt to suppress endogenous seed storage proteins by RNA interference (RNAi) to develop rice seeds as a more efficient protein expression system. In this study, human growth hormone (hGH) was expressed in transgenic rice seeds using an endosperm-specific promoter from a 10 kDa rice prolamin gene. In addition, an RNAi cassette for reduction of endogenous storage protein expressions was inserted into the hGH expression construct. Using this system, the expression levels of 13 kDa prolamin and glutelin were effectively suppressed and hGH polypeptides accumulated to 470 μg/g dry weight at the maximum level in transgenic rice seeds. These results suggest that the suppression of endogenous protein gene expression by RNAi could be of great utility for increasing transgene products.     

Secretion of human growth hormone by the food-grade bacterium Staphylococcus carnosus requires a propeptide irrespective of the signal peptide used

Staphylococcal exoproteins can be divided into two groups. One group comprises proteins bearing only a signal peptide, the other group requires an additional propeptide for secretion. The secretion signals of the propeptide-requiring lipase from Staphylococcus hyicus (Lip) have been frequently used to produce recombinant secretory proteins in the food-grade species Staphylococcus carnosus. However, it has been unclear whether recombinant proteins can be secreted using signal peptides of staphylococcal proteins without propeptide. The human growth hormone protein (hGH) was fused to various staphylococcal secretion signals of proteins without propeptide (Seb, SceA, and SceB) and of proteins requiring a propeptide (lipase, lysostaphin, and glycerol ester hydrolase). Secretory hGH was efficiently produced by S. carnosus after fusion with any propeptide-containing secretion signal, whereas precursor proteins were retained in the cells when only a signal peptide was used. Addition of the first six amino acid residues of mature SceA to the signal peptide did also not lead to secretion of hGH. It was concluded that the properties of the mature protein domains determine whether a propeptide is required for secretion or not. The Lip propeptide could be efficiently removed from hGH after introduction of an enterokinase cleavage site between the two protein domains.     

MALDI‐TOF characterization of hGH1 produced by hairy root cultures of Brassica oleracea var. italica grown in an airlift with mesh bioreactor

Expression systems based on plant cells, tissue, and organ cultures have been investigated as an alternative for production of human therapeutic proteins in bioreactors. In this work, hairy root cultures of Brassica oleracea var. italica (broccoli) were established in an airlift with mesh bioreactor to produce isoform 1 of the human growth hormone (hGH1) as a model therapeutic protein. The hGH1 cDNA was cloned into the pCAMBIA1105.1 binary vector to induce hairy roots in hypocotyls of broccoli plantlets via Agrobacterium rhizogenes. Most of the infected plantlets (90%) developed hairy roots when inoculated before the appearance of true leaves, and keeping the emerging roots attached to hypocotyl explants during transfer to solid Schenk and Hildebrandt medium. The incorporation of the cDNA into the hairy root genome was confirmed by PCR amplification from genomic DNA. The expression and structure of the transgenic hGH1 was assessed by ELISA, western blot, and MALDITOF‐MS analysis of the purified protein extracted from the biomass of hairy roots cultivated in bioreactor for 24 days. Production of hGH1 was 5.1 ± 0.42 µg/g dry weight (DW) for flask cultures, and 7.8 ± 0.3 µg/g DW for bioreactor, with productivity of 0.68 ± 0.05 and 1.5 ± 0.06 µg/g DW*days, respectively, indicating that the production of hGH1 was not affected by the growth rate, but might be affected by the culture system. These results demonstrate that hairy root cultures of broccoli have potential as an alternative expression system for production of hGH1, and might also be useful for production of other therapeutic proteins. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:161–171, 2014     

Effects of glucose on the production of recombinant protein C in mammalian cell culture

Effects of glucose on a cultured Chinese hamster ovary cell line producing recombinant human protein C were investigated. After the recombinant cells reached confluency, they were maintained in the medium containing 10% serum and different levels of glucose in either batch or daily-exchange mode. High concentrations of glucose to the cultures yielded higher cell densities. Daily exchanges of media produced higher cell densities than the corresponding batch culture. Total protein C production per cell decreased with time in batch culture, in accordance with the declined glucose metabolism. Supplementation of the media with high levels of glucose diminished both the expression and gamma-carboxylation activities of the recombinant cells. Production of protein C persisted in daily-exchange culture, resulting in a constant production rate of protein C. In this case again, glucose reduced the specific productivity of recombinant protein C. An apparent glucose inhibition constant was determined to be 0.11 mg/mL by Dixon plots. The ability to gamma-carboxylate recombinant protein C was also impaired at the highest level of glucose. From these results, a strategy to maximize recombinant protein C productivity is discussed.     

High-level secretion of the extracellular domain of the human growth hormone receptor using a baculovirus system

A chemically synthesized gene (hGHR-ED) coding for the extracellular domain (ED) of the human growth hormone (hGH) receptor (hGHR) was inserted into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. Spodoptera frugiperda cells infected with the recombinant virus secreted a protein with hGH-binding activity into the medium. The secreted 35-kDa protein was purified to near homogeneity. The purified protein exhibited a high binding affinity (Kd = 0.2-0.3 nM) to hGH. The highest cell production capability was estimated at more than 10-20 micrograms hGHR-ED/ml of culture. The inhibition of the hGHR-ED secretion by treatment with tunicamycin suggests that glycosylation is important for secretion.     

Surface Display of Human Growth Hormone on Bacillus subtilis Spores for Oral Administration

Human growth hormone (hGH) is the major and important hormone component of human being. At present, hGH for clinical uses is mostly produced in Escherichia coli, which requires costly denaturation and refolding to recover functionality. To obtain long-term bioactive hormone, we used hGH as a foreign gene and constructed a recombinant plasmid pJS700-hGH which carries a recombinant gene cotC-hgh with an enterokinase site under the control of cotC promoter. Plasmid pJS700-hGH was transformed into Bacillus subtilis by double crossover and an amylase-inactivated mutant was produced. After spore formation, Western blot and fluorescence immunoassay were used to monitor hGH surface expression on spores. Oral administration to silkworm with spores displaying hGH further showed that the recombinant spores may have potential ability to be digested and absorbed into the silkworm’s hemolymph due to both the resistant characters of spores and the addition of enterokinase site.