Production Of Mammalian Proteins Research Articles

Quantitative evaluation of mammalian skeletal muscle as a heterologous protein expression system

The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the fluorescent protein EGFP as a model, it is shown that muscle fibers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of ∼1 mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement.

Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli

The implementation of efficient technologies for the production of recombinant mammalian proteins remains an outstanding challenge in many structural and functional genomics programs. We have developed a new method for rapid identification of soluble protein expression in E. coli, based on a separation of soluble protein from inclusion bodies by a filtration step at the colony level. The colony filtration (CoFi) blot is very well suited to screen libraries, and in the present work we used it to screen a deletion mutagenesis library.

Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression

BackgroundIn the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44), kinases (EGFR-cytoplasmic domain, CDK2 and 4), proteases (MMP1, CASP2), signal transduction proteins (GRB2, RAF1, HRAS) and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX). Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study.ResultsSeveral protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY), or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx) and maltose binding protein (MBP) were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions.ConclusionsBy analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases proteins may be truncated to minimise molecular weight and the numbers of contiguous hydrophobic amino acids and low complexity regions to aid soluble expression in E. coli.

Secretion of human proteins from yeast: stimulation by duplication of polyubiquitin and protein disulfide isomerase genes in Kluyveromyces lactis

The efficiency of secreted production of mammalian proteins from yeasts remains unpredictably variable, depending on each protein. On the hypothesis that the control of protein conformation during protein translocation is the bottleneck in many cases, we examined the effects of an increased dosage of the genes coding for protein disulfide isomerase and of polyubiquitin on the secretion of two human proteins, serumalbumin and interleukin 1β. The yeast Kluyveromyces lactis was used as a production host. Duplication of either one of these genes had a strong stimulating effect on the production of the highly disulfide-bonded serumalbumin, but not of interleukin 1β.

Characterization of the post-translational biochemical processing of human lactoferrin expressed in transgenic tobacco

During the last couple of years it has been shown that transgenic plants are suitable host organisms for the production of recombinant mammalian proteins of therapeutic interest. Using transgenic tobacco plants as an experimental system we demonstrated that human lactoferrin, an iron-binding glycoprotein, is an interesting target molecule for this production system. Nonoptimized expression/extraction levels of up to 0.3% have so far been obtained. The recombinant human lactoferrin possesses similar molecular mass to native human milk lactoferrin but differs by its carbohydrate moiety. As this difference in glycosylation may represent a limitation in the use of lactoferrin for clinical applications, we are currently pursuing our research by expressing lactoferrin in other agronomic species and by modifying the glycosyltransferase equipment of the plant cell.

The hsp150 delta-carrier confers secretion competence to the rat nerve growth factor receptor ectodomain in Saccharomyces cerevisiae.

When the extracellular domain of rat low-affinity nerve growth factor receptor (NGFRe) was synthesized in Saccharomyces cerevisiae with the signal peptide of invertase, NGFRe was translocated to the endoplasmic reticulum (ER) and retained there. However, when NGFRe was fused to the C-terminus of the hsp150 delta-carrier, the hsp150 delta-NGFRe fusion protein was efficiently secreted to the growth medium with no apparent retention in the ER. The NGFRe portion was disulphide-bonded and its single N-glycosylation site was occupied. The hsp150 delta-carrier is an N-terminal signal peptide-containing fragment of a yeast secretory glycoprotein. Hsp150 delta-NGFRe, harvested from the culture medium, inhibited the cross-linking of [125I]NGF to authentic NGFR on the surface of human melanoma cells. Moreover, [125I]NGF could be chemically cross-linked to secretory hsp150 delta-NGFRe, suggesting that the NGFRe portion had adopted a ligand-binding conformation. However, inhibition of the cross-linking by unlabelled NGF was less effective than in the case of the authentic receptor. The hsp150 delta-carrier may have potential in the production of mammalian proteins, which require elaborate folding and disulphide formation in the ER.

High-level expression of soluble heterologous proteins in the cytoplasm of Escherichia coli by fusion to the bacteriophage lambda head protein D.

The bacteriophage Lambda head protein D (gpD) is a small major capsid protein (110aa; 11.6kDa; pI=5.68, devoid of cysteine residues) that is essential for stable head morphogenesis. We found that a His-tagged derivative of gpD (gpHD) is a monomeric protein with efficient expression properties and high resistance towards thermally induced irreversible aggregation. In addition, gpHD can be used as a fusion partner for high-level expression of soluble heterologous proteins in the cytoplasm of Escherichia coli. Its broad utility is illustrated by the production of various mammalian proteins by fusion to its C-terminus. As a fusion partner, gpHD is thought to mediate optimal translation initiation while reducing inclusion body formation and protein degradation. In addition, it provides a His-tag for simple purification. gpHD may act as a 'cytoplasmic anchor' by keeping its unfolded fusion partner in solution, thereby providing more time for proper folding. An ever-increasing number of open reading frames (ORFs) are being identified in the various genome sequencing programs. gpHD has the potential to be harnessed for the development of highly efficient cytoplasmic expression systems that might contribute to the production and characterization of these novel polypeptides. Protein D is also an established fusion partner for phage display. It thus presents the attractive opportunity of coupling the selection of heterologous proteins from a phage library to their subsequent high-level expression.

Use of the Glyceraldehyde-3-phosphate Dehydrogenase Promoter for Production of Functional Mammalian Membrane Transport Proteins in the YeastPichia pastoris

The promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (PGAP) was employed to produce the mammalian peptide transporters hPEPT1 and rPEPT2 as models for polytopic transmembrane proteins in the methylotrophic yeastPrichia pastoris.Cells of a recombinant renal peptide transporter (rPEPT2) clone produced constitutively the functional carrier protein. The level of functional expression of rPEPT2 with PGAPvaried depending on the carbon source used for cell growth, but was up to five times higher than that obtained with the commonly employed inducible alcohol oxidase 1 promoter (PAOX1). Similar results were obtained for the expression level of the human intestinal peptide transporter hPEPT1 controlled by either PGAPor PAOX1. Therefore, the PGAPseems to be an attractive alternative to PAOX1for generation of transgenicP. pastoriscells expressing functional mammalian membrane transport proteins at high levels.

Optimization of transient gene expression in mammalian cells and potential for scale-up using flow electroporation.

The goals of this study were to identify mammalian cell lines which could be efficiently transiently-transfected and scaled-up for protein production. The transfection efficiencies of eight cell lines (NSO, NSO-TAg, CV-1, COS-7, CHO, CHO-TAg, HEK 293, and 293-EBNA) were measured using electroporation for DNA delivery and green fluorescent protein (Evans, 1996) as the reporter gene. In addition, we have evaluated the effects of stable expression of viral proteins, cell cycle manipulation, and butyrate post-treatment in small scale experiments. The cell lines varied widely in their GFP transfection efficiencies. Stable expression of simian virus 40 large T-antigen or Epstein Barr nuclear antigen failed to significantly increase transfection efficiency above that seen in the parental lines. Aphidicolin (a DNA polymerase inhibitor), which blocked cells from S or G2/M, brought about an increase in transfection efficiency in two cell lines. The primary effect of butyrate (a histone deacetylase inhibitor) post-treatment was an increased intensity of the fluorescent signal of green fluorescent protein, as measured by flow cytometry (1.0 to 4.2-fold, depending on the cell line). The combined use of aphidicolin pretreatment followed by butyrate treatment post- electroporation yielded increases in fluorescence intensities ranging from 0.9 to 6.8-fold. Based on their high transfection efficiencies in small scale experiments, rapid growth, and ability to grow in suspension culture, CHO, CHO-TAg, and 293-EBNA were selected to assess the feasibility of using flow electroporation for large-scale transfections. Using secreted placental alkaline phosphatase as a reporter, 293-EBNA cells produced the highest protein levels in both the presence and absence of butyrate. These data indicate that flow electroporation provides an efficient method of DNA delivery into large numbers of cells for mammalian protein production.

Human prion proteins expressed in Escherichia coli and purified by high-affinity column refolding

An efficient method is presented for the production of intact mammalian prion proteins and partial sequences thereof. As an illustration we describe the production of polypeptides comprising residues 23–231, 81–231, 90–231 and 121–231 of the human prion protein ( hPrP) 1 Sequence positions according to Syrian hamster PrP [1]. 1 . Polypeptides were expressed as histidine tail fusion proteins into inclusion bodies in the cytoplasm of Escherichia coli and refolded and oxidized while N-terminally immobilized on a nickel-NTA agarose resin. This `high-affinity column refolding' facilitates the preparation of prion proteins by preventing protein aggregation and intermolecular disulfide formation. After elution from the resin the histidine tail can be removed using thrombin without cleaving the prion protein polypeptide chain. The same protocol as used here for hPrP has been successfully applied with bovine and murine prion proteins. The protein preparations are stable for weeks at room temperature in concentrated solution and are thus suitable for detailed structural studies. Preliminary biophysical characterization of hPrP(23–231) suggests that the C-terminal half of the polypeptide chain forms a well-structured globular domain, and that the N-terminal half does not form extensive regular secondary structures.

The hsp150Δ-carrier confers secretion competence to the rat nerve growth factor receptor ectodomain in Saccharomyces cerevisiae

When the extracellular domain of rat low-affinity nerve growth factor receptor (NGFRe) was synthesized in Saccharomyces cerevisiae with the signal peptide of invertase, NGFRe was translocated to the endoplasmic reticulum (ER) and retained there. However, when NGFRe was fused to the C-terminus of the hsp150Δ-carrier, the hsp150Δ–NGFRe fusion protein was efficiently secreted to the growth medium with no apparent retention in the ER. The NGFRe portion was disulphide-bonded and its single N-glycosylation site was occupied. The hsp150Δ-carrier is an N-terminal signal peptide-containing fragment of a yeast secretory glycoprotein. Hsp150Δ–NGFRe, harvested from the culture medium, inhibited the cross-linking of [125I]NGF to authentic NGFR on the surface of human melanoma cells. Moreover, [125I]NGF could be chemically cross-linked to secretory hsp150Δ–NGFRe, suggesting that the NGFRe portion had adopted a ligand-binding conformation. However, inhibition of the cross-linking by unlabelled NGF was less effective than in the case of the authentic receptor. The hsp150Δ-carrier may have potential in the production of mammalian proteins, which require elaborate folding and disulphide formation in the ER.

Enhanced Recovery of a Secreted Mammalian Protein from Suspension Culture of Genetically Modified Tobacco Cells

Increasing the level of recovery of mammalian proteins secreted by a genetically modifiedNicotiana tabacumwas explored in suspension culture. As a model protein system, a mouse monoclonal antibody heavy chain γ (MAb HC) with an antigen specificity forp-azophenylarsonate was used. Consistent with findings for other plant cell suspension culture systems expressing proteins with mammalian leader sequences, the synthesized mouse MAb HC was secreted through the plasma membrane. In addition, the majority of the MAb HC was also secreted through the cell wall into the growth medium. However, efficient recovery of the protein was only possible when the protein stabilizing agent, polyvinylpyrrolidone (PVP) was present in the plant cell growth medium. The presence of PVP increased the recovered concentration of secreted protein 35-fold from 0.010 to 0.36 μg protein/ml culture medium. Biological activity of the ∼50-kDa MAb HC polypeptide was demonstrated by arsonate affinity matrix binding as detemined by Western blot analysis. In addition to antigen binding activity, the secreted protein also exhibited reactivity to protein G, a protein which specifically binds mouse IgG. These findings are important because they demonstrate that culture conditions can significantly influence the concentration of a biologically active foreign protein secreted from plant cells into the media of suspension cultures. The ability to increase the efficiency of mammalian protein production in plant suspension culture systems should provide significant advantage over protein production in intact transgenic plants which require cultivation, harvesting, and expensive extraction procedures to obtain nonsecreted foreign proteins.

Myeloma based expression system for production of large mammalian proteins

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Mammalian recombinant proteins: vectors and expression systems.

The determination of the biological properties and physiological significance of proteins is often hampered by the low level at which their genes are naturally expresSed. The therapeutic use of mammalian proteins is also limited by the availability of authentic material. A number of expression systems are now available for the high level production of mammalian proteins and, in some cases, these have been used to generate material of therapeutic use. Proteins such as human growth hormone, insulin, erythropoietin, tissue plasminogen activator, interleukin (IL)-2 and the interferons (IFNs) are examples of human therapeutic agents produced using heterologous expression systems. Although efficient expression can be achieved in many cases, there is a continued need for the improvement and refinement of expression systems to allow for the rapid production of authentic recombinant proteins including those that are lethal to host cells. To that end, a variety of expression systems have been devised and modified, each with distinct advantages and disadvantages. In order to be certain of producing bioactive proteins, it is essential that these alternative approaches are tried, as no one system can fi.tlfil all the requirements for successful production of mammalian proteins. In this review, I will summarize the main advances in the past year or two in the development of expression systems for mammalian recombinant proteins.

Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates.

Purification of Proteins and the Disruption of Microbial Cells

AbstractIntracellular proteins with catalytic or biological activity are of growing importance for developments in enzyme technology, as well as for the production of mammalian proteins by recombinant‐DNA technology. The release of these proteins from microorganisms is an important unit operation, as it is the first step in their isolation.Gram‐scale disruption of microorganisms can be performed by a variety of established methods based on chemical, enzymatic, physical, or mechanical principles. For the large scale disruption of microorganisms, mechanical disintegrators, such as high‐speed agitator bead mills or high‐pressure industrial homogenizers, are commonly employed. Both types of equipment were designed originally for other tasks; in the paint industry or in the milk industry, respectively. Therefore, it appeared necessary and possible to improve design and performance for the application in cell disintegration. The goal is a uniform exposure of the microbial cells and a minimal exposure of solubilized protein to high shear forces, in order to obtain high yields and to avoid the generation of too small cell wall fragments, which are difficult to separate.Both types of machines have been investigated for the disintegration of different microbial cells and the influence of the operating parameters analyzed on protein solubilization and enzyme yield. We will summarize the state of the art and discuss new data to illustrate trends in process development.

Low Molecular Weight High–Mannose Type Glycans in a Secreted Protein of the Filamentous Fungus Trichoderma Reesei

With the object of obtaining information about the suitability of filamentous fungi for production of heterogeneous mammalian proteins, we have characterized the normal glycosylation pattern of CBH I, the major glycoprotein secreted by Trichoderma reesei. The protein–bound glycans of CBH I were studied using a variety of separation techniques to analyze the products of specific glycosidase digestions and chemical degradation procedures. CBH I contains both O–glycosidic and N–glycosidic glycans. The O–glycosidic glycans consist of one to four hexose residues which are mostly mannose. The structures of the N–glycosidic glycans are (Man)5(GlcNAc)2 and (Man)9(GlcNAc)2. Thus it may be possible to use T. reesei as a host for the production of heterologous glycoproteins of animal origin, and to take advantage of its remarkable secretory capacity.