Production of Recombinant Human Ceruloplasmin: Improvements and Perspectives.
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ABSTRACT:
SUBMITTER: Bonaccorsi di Patti MC
PROVIDER: S-EPMC8347646 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Salicylate can be biosynthesized from the common metabolic intermediate shikimate and has found applications in pharmaceuticals and in the bioplastics industry. While much metabolic engineering work focused on the shikimate pathway has led to the biosynthesis of a variety of aromatic compounds, little is known about how the relative expression levels of pathway components influence salicylate biosynthesis. Furthermore, some host strain gene deletions that improve salicylate production may be impossible to predict. Here, a salicylate-responsive transcription factor was used to optimize the expression levels of shikimate/salicylate pathway genes in recombinant E. coli, and to screen a chromosomal transposon insertion library for improved salicylate production. RESULTS:A high-throughput colony screen was first developed based on a previously designed salicylate-responsive variant of the E. coli AraC regulatory protein ("AraC-SA"). Next, a combinatorial library was constructed comprising a series of ribosome binding site sequences corresponding to a range of predicted protein translation initiation rates, for each of six pathway genes (>?38,000 strain candidates). Screening for improved salicylate production allowed for the rapid identification of optimal gene expression patterns, conferring up to 123% improved production of salicylate in shake-flask culture. Finally, transposon mutagenesis and screening revealed that deletion of rnd (encoding RNase D) from the host chromosome further improved salicylate production by 27%. CONCLUSIONS:These results demonstrate the effectiveness of the salicylate sensor-based screening platform to rapidly identify beneficial gene expression patterns and gene knockout targets for improving production. Such customized high-throughput tools complement other cell factory engineering strategies. This approach can be generalized for the production of other shikimate-derived compounds.
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Project description:We have previously described the development and implementation of a strategy for production of recombinant polyclonal antibodies (rpAb) in single batches employing CHO cells generated by site-specific integration, the Sympress I technology. The Sympress I technology is implemented at industrial scale, supporting a phase II clinical development program. Production of recombinant proteins by site-specific integration, which is based on incorporation of a single copy of the gene of interest, makes the Sympress I technology best suited to support niche indications. To improve titers while maintaining a cost-efficient, highly reproducible single-batch manufacturing mode, we have evaluated a number of different approaches. The most successful results were obtained using random integration in a new producer cell termed ECHO, a CHO DG44 cell derivative engineered for improved productivity at Symphogen. This new expression process is termed the Sympress II technology. Here we describe proof-of-principle data demonstrating the feasibility of the Sympress II technology for single-batch rpAb manufacturing using two model systems each composed of six target-specific antibodies. The compositional stability and the batch-to-batch reproducibility of rpAb produced by the ECHO cells were at least as good as observed previously using site-specific integration technology. Furthermore, the new process had a significant titer increase.
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Project description:Various strategies of plant breeding have been attempted in order to improve the ethylene resistance of flowering ornamental plants. These approaches span from conventional techniques such as simple cross-pollination to new breeding techniques which modify the plants genetically such as precise genome-editing. The main strategies target the ethylene pathway directly; others focus on changing the ethylene pathway indirectly via pathways that are known to be antagonistic to the ethylene pathway, e.g. increasing cytokinin levels. Many of the known elements of the ethylene pathway have been addressed experimentally with the aim of modulating the overall response of the plant to ethylene. Elements of the ethylene pathway that appear particularly promising in this respect include ethylene receptors as ETR1, and transcription factors such as EIN3. Both direct and indirect approaches seem to be successful, nevertheless, although genetic transformation using recombinant DNA has the ability to save much time in the breeding process, they are not readily used by breeders yet. This is primarily due to legislative issues, economic issues, difficulties of implementing this technology in some ornamental plants, as well as how these techniques are publically perceived, particularly in Europe. Recently, newer and more precise genome-editing techniques have become available and they are already being implemented in some crops. New breeding techniques may help change the current situation and pave the way toward a legal and public acceptance if products of these technologies are indistinguishable from plants obtained by conventional techniques.
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Project description:Multicopper oxidases (MCOs) are a large family of diverse enzymes found in both eukaryotes and prokaryotes that couple one-electron oxidations of various substrates to the four-electron reduction of O2 to H2O, functioning through a set of metallocofactors consisting of one type 1 copper (T1 Cu) and one trinuclear copper cluster (TNC). Human serum ceruloplasmin (Cp) is a unique member of MCOs composed of six cupredoxin domains and harbors six Cu ions arranged as three T1 Cu and one TNC. The native substrate of Cp is Fe2+. It is an essential ferroxidase critical for iron homeostasis and is closely associated with metal-mediated diseases and metal neurotoxicity. In human serum, Cp operates under substrate-limiting low [Fe2+] but high [O2] conditions, implying the possible involvement of partially reduced intermediates in Cp catalysis. In this work, we studied for the first time Cp reactivities at defined partially reduced states and discovered a tyrosine radical weakly magnetically coupled to the native intermediate (NI) of the TNC via a hydrogen bond. Our results lead to a new hypothesis that human iron transport is regulated as the paired transfer of iron from ferroportin to Cp to transferrin, and the tyrosine residue in Cp acts as a gate to avoid reactive oxygen species (ROS) formation when Fe2+ delivery is dysregulated.
| S-EPMC7596862 | biostudies-literature
Project description:This study was carried out to express human epidermal growth factor (hEGF) in Pichia pastoris GS115. For this aim, the hEGF gene was cloned into the pPIC9K expression vector, and then integrated into P. pastoris by electroporation. ELISA-based assay showed that the amount of hEGF secreted into the medium can be affected by the fermentation conditions especially by culture medium, pH and temperature. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. The highest amount of hEGF with an average yield of 2.27?g/mL was obtained through an induction of the culture with 0.5% (v/v) methanol for 60h. The artificial neural network (ANN) analysis revealed that changes in both pH and temperature significantly affected the hEGF production with the pH change had slightly higher impact on hEGF production than variations in the temperature.
| S-EPMC5470459 | biostudies-literature
Project description:BACKGROUND: Annexin V, a 35.8 kDa intracellular protein, is a Ca?²-dependent phospholipid binding protein with high affinity to phosphatidylserine (PS), which is a well-known hallmark of apoptosis. Annexin V is a sensitive probe for PS exposure upon the cell membrane, and used for detection of apoptotic cells both in vivo and in vitro. Large-scale production of recombinant human annexin V is worth optimization, because of its wide use in nuclear medicine, radiolabeled with (99m)Tc, for the evaluation of cancer chemotherapy treatments, and its use in identification of apoptotic cells in histologic studies. Here we describe the high-yield production of a tag-free version of human annexin V recombinant protein by linear fed-batch cultivation in a bioreactor. RESULTS: We cloned the human ANXA5 coding sequence into the pET-30a (+) expression vector and expressed rhANXA5 in batch and fed-batch cultures. Using E. coli BL21 (DE3) in a semi-defined medium at 37°C, pH 7 in fed-batch cultures, we obtained a 45-fold increase in biomass production, respective to shaker cultivations. We developed a single-step protocol for rhANXA5 purification using a strong anion-exchange column (MonoQ HR16/10). Using these procedures, we obtained 28.5 mg of homogeneous, nontagged and biologically functional human annexin V recombinant protein from 3 g wet weight of bacterial cells from bioreactor cultures. The identity and molecular mass of rhANXA5 was confirmed by mass spectrometry. Moreover, the purified rhANXA5 protein was functionally evaluated in a FITC-annexin V binding experiment and the results demonstrated that rhANXA5 detected apoptotic cells similarly to a commercial kit. CONCLUSIONS: We describe a new fed-batch method to produce recombinant human annexin V in large scale, which may expand the commercial utilities for rhANXAV to applications such as in vivo imaging studies.
| S-EPMC4029966 | biostudies-literature
Project description:Methods for recombinant production of eukaryotic membrane proteins, yielding sufficient quantity and quality of protein for structural biology, remain a challenge. We describe here, expression and purification optimisation of the human SERCA2a cardiac isoform of Ca(2+) translocating ATPase, using Saccharomyces cerevisiae as the heterologous expression system of choice. Two different expression vectors were utilised, allowing expression of C-terminal fusion proteins with a biotinylation domain or a GFP- His8 tag. Solubilised membrane fractions containing the protein of interest were purified onto Streptavidin-Sepharose, Ni-NTA or Talon resin, depending on the fusion tag present. Biotinylated protein was detected using specific antibody directed against SERCA2 and, advantageously, GFP-His8 fusion protein was easily traced during the purification steps using in-gel fluorescence. Importantly, talon resin affinity purification proved more specific than Ni-NTA resin for the GFP-His8 tagged protein, providing better separation of oligomers present, during size exclusion chromatography. The optimised method for expression and purification of human cardiac SERCA2a reported herein, yields purified protein (> 90%) that displays a calcium-dependent thapsigargin-sensitive activity and is suitable for further biophysical, structural and physiological studies. This work provides support for the use of Saccharomyces cerevisiae as a suitable expression system for recombinant production of multi-domain eukaryotic membrane proteins.
| S-EPMC3741278 | biostudies-literature
Project description:There is a steady increasing demand for insulin worldwide. Current insulin manufacturing capacities can barely meet this increasing demand. The purpose of this study was to test the feasibility of producing human proinsulin in the milk of transgenic animals. Four lines of transgenic mice harboring a human insulin cDNA with expression driven by the goat ?-casein gene promoter were generated. The expression level of human proinsulin in milk was as high as 8.1 g/L. The expression of the transgene was only detected in the mammary gland during lactation, with higher levels at mid-lactation and lower levels at early and late lactation. The blood glucose and insulin levels and the major milk compositions were unchanged, and the transgenic animals had no apparent health defects. The mature insulin derived from the milk proinsulin retained its biological activity. In conclusion, our study provides supporting evidence to explore the production of high levels of human proinsulin in the milk of dairy animals.
| S-EPMC4179469 | biostudies-literature
Project description:BackgroundAn arbitrary set of 96 human proteins was selected and tested to set-up a fully automated protein production strategy, covering all steps from DNA preparation to protein purification and analysis. The target proteins are encoded by functionally uncharacterized open reading frames (ORF) identified by the German cDNA consortium. Fusion proteins were produced in E. coli with four different fusion tags and tested in five different purification strategies depending on the respective fusion tag. The automated strategy relies on standard liquid handling and clone picking equipment.ResultsA robust automated strategy for the production of recombinant human proteins in E. coli was established based on a set of four different protein expression vectors resulting in NusA/His, MBP/His, GST and His-tagged proteins. The yield of soluble fusion protein was correlated with the induction temperature and the respective fusion tag. NusA/His and MBP/His fusion proteins are best expressed at low temperature (25 degrees C), whereas the yield of soluble GST fusion proteins was higher when protein expression was induced at elevated temperature. In contrast, the induction of soluble His-tagged fusion proteins was independent of the temperature. Amylose was not found useful for affinity-purification of MBP/His fusion proteins in a high-throughput setting, and metal chelating chromatography is recommended instead.ConclusionSoluble fusion proteins can be produced in E. coli in sufficient qualities and microg/ml culture quantities for downstream applications like microarray-based assays, and studies on protein-protein interactions employing a fully automated protein expression and purification strategy. Future applications might include the optimization of experimental conditions for the large-scale production of soluble recombinant proteins from libraries of open reading frames.
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Project description:The production of recombinant proteins in plant systems is receiving wider attention. Indeed, various plant-produced pharmaceuticals have been shown to be biologically active. However, the production of human growth factors and cytokines in heterologous systems is still challenging because they often act as complex forms, such as homo- or hetero-dimers, and their production is tightly regulated in vivo. In this study, we demonstrated that the mature form of human TGFβ1 produced and purified from Nicotiana benthamiana shows biological activity in animal cells. To produce the mature form of TGFβ1, various recombinant genes containing the mature form of TGFβ1 were generated and produced in N. benthamiana. Of these, a recombinant construct, BiP:M:CBM3:LAP[C33S]:EK:TGFβ1, was expressed at a high level in N. benthamiana. Recombinant proteins were one-step purified using cellulose-binding module 3 (CBM3) as an affinity tag and microcrystalline cellulose (MCC) beads as a matrix. The TGFβ1 recombinant protein bound on MCC beads was proteolytically processed with enterokinase to separate mature TGFβ1. The mature TGFβ1 still associated with Latency Associated Protein, [LAP(C33S)] that had been immobilized on MCC beads was released by HCl treatment. Purified TGFβ1 activated TGFβ1-mediated signaling in the A549 cell line, thereby inducing phosphorylation of SMAD-2, the expression of ZEB-2 and SNAIL1, and the formation of a filopodia-like structure. Based on these results, we propose that active mature TGFβ1, one of the most challenging growth factors to produce in heterologous systems, can be produced from plants at a high degree of purity via a few steps.
| S-EPMC9197560 | biostudies-literature