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Heterologous Protein Production (heterologous + protein_production)
Selected AbstractsHeterologous Protein Production from the Inducible MET25 Promoter in Saccharomyces cerevisiaeBIOTECHNOLOGY PROGRESS, Issue 2 2005Steven P. Solow Heterologous protein production late in Saccharomyces cerevisiae fermentations is often desirable because it may help avoid the unintentional selection of more rapidly growing, non-protein-expressing cells or allow for the expression of toxic proteins. Here, we describe the use of the MET25 promoter for the production of human serum albumin (HSA) and HSA-fusion proteins in S. cerevisiae. In media lacking methionine, the MET25 promoter yielded high expression levels of HSA and HSA fused to human glucagon, human growth hormone, human interferon ,, and human interleukin-2. More importantly, we have shown that this system can be used to delay heterologous protein production until late log phase of the growth of the culture and does not require the addition of an exogenous inducer. [source] Non-invasive detection of the metabolic burden on recombinant microorganisms during fermentation processes,JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 8 2001Th Bachinger Abstract Heterologous protein production is an important source of therapeutic products. Optimisation of such bioprocesses by adjustment of the expression rate of the heterologous protein to the biosynthetic capacity of the cell metabolism would benefit from an online method for monitoring the metabolic burden. In this study we evaluated the use of a chemical multi-sensor array for this purpose. Fermentations with a recombinant Escherichia coli strain expressing human superoxide dismutase (rhSOD) were monitored by the sensor array. The results of isopropyl-thiogalactopyranoside (IPTG)-induced expression were compared with fermentations with a plasmid-free strain. The overproduction of rhSOD, imposing a high metabolic burden on the plasmid-carrying cells, was distinctly and reproducibly observed by the multi-sensor array. The potential of this non-invasive method of non-specific metabolic burden monitoring is demonstrated by the results of the study. © 2001 Society of Chemical Industry [source] KNQ1, a Kluyveromyces lactis gene encoding a transmembrane protein, may be involved in iron homeostasisFEMS YEAST RESEARCH, Issue 5 2007Emmanuela Marchi Abstract The original purpose of the experiments described in this article was to identify, in the biotechnologically important yeast Kluyveromyces lactis, gene(s) that are potentially involved in oxidative protein folding within the endoplasmic reticulum (ER), which often represents a bottleneck for heterologous protein production. Because treatment with the membrane-permeable reducing agent dithiothreitol inhibits disulfide bond formation and mimics the reducing effect that the normal transit of folding proteins has in the ER environment, the strategy was to search for genes that conferred higher levels of resistance to dithiothreitol when present in multiple copies. We identified a gene (KNQ1) encoding a drug efflux permease for several toxic compounds that in multiple copies conferred increased dithiothreitol resistance. However, the KNQ1 product is not involved in the excretion of dithiothreitol or in recombinant protein secretion. We generated a knq1 null mutant, and showed that both overexpression and deletion of the KNQ1 gene resulted in increased resistance to dithiothreitol. KNQ1 amplification and deletion resulted in enhanced transcription of iron transport genes, suggesting, for the membrane-associated protein Knq1p, a new, unexpected role in iron homeostasis on which dithiothreitol tolerance may depend. [source] Approaches to achieve high-level heterologous protein production in plantsPLANT BIOTECHNOLOGY JOURNAL, Issue 1 2007Stephen J. Streatfield Summary Plants offer an alternative to microbial fermentation and animal cell cultures for the production of recombinant proteins. For protein pharmaceuticals, plant systems are inherently safer than native and even recombinant animal sources. In addition, post-translational modifications, such as glycosylation, which cannot be achieved with bacterial fermentation, can be accomplished using plants. The main advantage foreseen for plant systems is reduced production costs. Plants should have a particular advantage for proteins produced in bulk, such as industrial enzymes, for which product pricing is low. In addition, edible plant tissues are well suited to the expression of vaccine antigens and pharmaceuticals for oral delivery. Three approaches have been followed to express recombinant proteins in plants: expression from the plant nuclear genome; expression from the plastid genome; and expression from plant tissues carrying recombinant plant viral sequences. The most important factor in moving plant-produced heterologous proteins from developmental research to commercial products is to ensure competitive production costs, and the best way to achieve this is to boost expression. Thus, considerable research effort has been made to increase the amount of recombinant protein produced in plants. This research includes molecular technologies to increase replication, to boost transcription, to direct transcription in tissues suited for protein accumulation, to stabilize transcripts, to optimize translation, to target proteins to subcellular locations optimal for their accumulation, and to engineer proteins to stabilize them. Other methods include plant breeding to increase transgene copy number and to utilize germplasm suited to protein accumulation. Large-scale commercialization of plant-produced recombinant proteins will require a combination of these technologies. [source] Characteristics of Saccharomyces cerevisiae gal1, and gal1,hxk2, mutants expressing recombinant proteins from the GAL promoterBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2005Hyun Ah Kang Abstract Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1, mutant strain was constructed and its induction kinetics investigated. As expected, the gal1, strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.05,0.1 g/L). However, the gal1, strain produced much more ethanol, in a complex medium containing glucose, than the GAL1 strain. To improve the physiological properties of the gal1, mutant strain as a host for heterologous protein production, a null mutation of either MIG1 or HXK2 was introduced into the gal1, mutant strain, generating gal1,mig1, and gal1,hxk2, double strains. The gal1,hxk2, strain showed a decreased rate of ethanol synthesis, with an accelerated rate of ethanol consumption, compared to the gal1, strain, whereas the gal1,mig1, strain showed similar patterns to the gal1, strain. Furthermore, the gal1,hxk2, strain secreted much more recombinant proteins (HSA and HSA fusion proteins) than the other strains. The results suggest that the gal1,hxk2, strain would be useful for the large-scale production of heterologous proteins from the GAL10 promoter in S. cerevisiae. © 2005 Wiley Periodicals, Inc. [source] Novel CNBP- and La-based translation control systems for mammalian cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2003Stefan Schlatter Abstract Throughout the development of Xenopus, production of ribosomal proteins (rp) is regulated at the translational level. Translation control is mediated by a terminal oligopyrimidine element (TOP) present in the 5, untranslated region (UTR) of rp -encoding mRNAs. TOP elements adopt a specific secondary structure that prevents ribosome-binding and translation-initiation of rp -encoding mRNAs. However, binding of CNBP (cellular nucleic acid binding protein) or La proteins to the TOP hairpin structure abolishes the TOP-mediated transcription block and induces rp production. Based on the specific CNBP-TOP/La-TOP interactions we have designed a translation control system (TCS) for conditional as well as adjustable translation of desired transgene mRNAs in mammalian cells. The generic TCS configuration consists of a plasmid encoding CNBP or La under control of the tetracycline-responsive expression system (TETOFF) and a target expression vector containing a TOP module between a constitutive PSV40 promoter and the human model product gene SEAP (human secreted alkaline phosphatase) (PSV40 -TOP- SEAP -pA). The TCS technology showed excellent SEAP regulation profiles in transgenic Chinese hamster ovary (CHO) cells. Alternatively to CNBP and La, TOP-mediated translation control can also be adjusted by artificial phosphorothioate anti-TOP oligodeoxynucleotides. Confocal laser-scanning microscopy demonstrated cellular uptake of FITC-labeled oligodeoxynucleotides and their localization in perinuclear organelles within 24 hours. Besides their TOP-based translation-controlling capacity, CNBP and La were also shown to increase cap-independent translation from polioviral internal ribosomal entry sites (IRES) and La alone to boost cap-dependent translation initiation. CNBP and La exemplify for the first time the potential of RNA-binding proteins to exert translation control of desired transgenes and to increase heterologous protein production in mammalian cells. We expect both of these assets to advance current gene therapy and biopharmaceutical manufacturing strategies. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 1,12, 2003. [source] Real-time PCR-based determination of gene copy numbers in Pichia pastorisBIOTECHNOLOGY JOURNAL, Issue 4 2010Sandra Abad Abstract Pichia pastoris is a preferred host for heterologous protein production. Expression cassettes are usually integrated into the genome of this methylotrophic yeast. This manuscript describes a method for fast and reliable gene copy number determinations for P. pastoris expression strains. We believe that gene copy number determinations are important for all researchers working with P. pastoris and also many other research groups using similar gene integration techniques for the transformation of other yeasts. The described method uses real-time PCR to quantify the integrated expression cassettes. Similar methods were employed previously for other host systems such as animal and plant cells but no such method comparing different detection methods and describing details for yeast analysis by quantitative PCR is known to us, especially for methylotrophic yeasts such as P. pastoris. Neglecting gene copy numbers can easily lead to false interpretations of experimental results from codon optimization or promoter studies and co-expression of helper proteins as demonstrated in an application example, which is also described here. [source] Expression system for recombinant human growth hormone production from Bacillus subtilisBIOTECHNOLOGY PROGRESS, Issue 1 2009Tunçer H. Özdamar Abstract We demonstrate for the first time, an expression system mimicking serine alkaline protease synthesis and secretion, producing native form of human growth hormone (hGH) from Bacillus subtilis. A hybrid-gene of two DNA fragments, i.e., signal (pre- ) DNA sequence of B. licheniformis serine alkaline protease gene (subC) and cDNA encoding hGH, were cloned into pMK4 and expressed under deg -promoter in B. subtilis. Recombinant-hGH (rhGH) produced by B. subtilis carrying pMK4::pre(subC)::hGH was secreted. N-terminal sequence and mass spectrometry analyses of rhGH confirm the mature hGH sequence, and indicate that the signal peptide was properly processed by B. subtilis signal-peptidase. The highest rhGH concentration was obtained at t = 32 h as CrhGH = 70 mg L,1 with a product yield on substrate YrhGH/S = 9 g kg,1, in a glucose based defined medium. Fermentation characteristics and influence of hGH gene on the rhGH production were investigated by comparing B. subtilis carrying pMK4::pre(subC)::hGH with that of carrying merely pMK4. Excreted organic-acid concentrations were higher by B. subtilis carrying pMK4::pre(subC)::hGH, whereas excreted amino-acid concentrations were higher by B. subtilis carrying pMK4. The approach developed is expected to be applicable to the design of expression systems for heterologous protein production from Bacillus species. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Heterologous Protein Production from the Inducible MET25 Promoter in Saccharomyces cerevisiaeBIOTECHNOLOGY PROGRESS, Issue 2 2005Steven P. Solow Heterologous protein production late in Saccharomyces cerevisiae fermentations is often desirable because it may help avoid the unintentional selection of more rapidly growing, non-protein-expressing cells or allow for the expression of toxic proteins. Here, we describe the use of the MET25 promoter for the production of human serum albumin (HSA) and HSA-fusion proteins in S. cerevisiae. In media lacking methionine, the MET25 promoter yielded high expression levels of HSA and HSA fused to human glucagon, human growth hormone, human interferon ,, and human interleukin-2. More importantly, we have shown that this system can be used to delay heterologous protein production until late log phase of the growth of the culture and does not require the addition of an exogenous inducer. [source] | ||||||||||