Production Of Milligram Research Articles

DNA-catalyzed efficient production of single-stranded DNA nanostructures

<h2>Summary</h2> Single-stranded (ss) DNA nanostructures (DNs), created by unimolecular folding of kilo-based ssDNA, represent a new class of DNA constructs suitable for biomaterial and biomedical applications. Here, using DNA-cleaving DNA enzymes (deoxyribozymes), we present a strategy for the efficient and cost-effective production of milligrams of ssDNs. We designed "genes" of ssDNs flanked by self-cleaving deoxyribozymes and amplified the genes in the single-stranded form. Upon autoprocessing, the resulting amplicons release a quadrillion copies of the ssDNA that self-folds into the desired structures. By engineering <i>trans</i>-acting deoxyribozymes, we showed that several ssDNs of different shapes can be encoded and amplified in tandem with controllable release of specific shapes from the amplicons, thereby providing a convenient yet economical way to safely store "genetic codes" of nanostructures on DNA for selective mass production on demand. We demonstrated ssDNs' ability to assist liposome sorting and expect the approach will promote the development of ssDNs' applications in broad areas.

On-resin strategy to label α-conotoxins: Cy5-RgIA, a potent α9α10 nicotinic acetylcholine receptor imaging probe.

In-solution conjugation is the most commonly used strategy to label peptides and proteins with fluorophores. However, lack of site-specific control and high costs of fluorophores are recognised limitations of this approach. Here, we established facile access to grams of Cy5-COOH via a two-step synthetic route, demonstrated that Cy5 is stable to HF treatment and therefore compatible with Boc-SPPS, and coupled Cy5 to the N-terminus of α-conotoxin RgIA while still attached to the resin. Folding of the two-disulfide containing Cy5-RgIA benefitted from the hydrophobic nature of Cy5 resulting in only the globular disulfide bond isomer. In contrast, wild-type α-RgIA folded into the inactive ribbon and bioactive globular isomer under the same conditions. Labelled α-RgIA retained its ability to inhibit acetylcholine(100 μM)-evoked current reversibly with an IC50 of 5.0 nM (Hill coefficient = 1.7) for α-RgIA and an IC50 of 1.6 (Hill coefficient = 1.2) for Cy5-RgIA at the α9α10 nicotinic acetylcholine receptors (nAChRs) heterologeously expressed in Xenopus oocytes. Cy5-RgIA was then used to successfully visualise nAChRs in RAW264.7 mouse macrophage cell line. This work introduced not only a new and valuable nAChR probe, but also a new versatile synthetic strategy that facilitates production of milligram to gram quantities of fluorophore-labelled peptides at low cost, which is often required for in vivo experiments. The strategy is compatible with Boc- and Fmoc-chemistry, allows for site-specific labelling of free amines anywhere in the peptide sequence, and can also be used for the introduction of Cy3/Cy5 FRET pairs.

DNA Catalyzed Efficient Production of Single-Stranded DNA Nanostructures

Single-stranded (ss) DNA nanostructures, created by unimolecular folding of kilo-based ssDNA, represent a novel class of DNA constructs suitable for biomaterial and biomedical applications. Here, using DNA-cleaving DNA enzymes (deoxyribozymes), we present a strategy for efficient and cost-effective production of milligrams of ssDNA nanostructures. We designed “genes” of ssDNA nanostructures flanked by self-cleaving deoxyribozymes and amplified the genes in the single-stranded form. Upon autoprocessing, the resulting amplicons release quadrillion copies of the ssDNA that self-folds into the desired structures. By engineering trans-acting deoxyribozymes, we showed that several ssDNA nanostructures of different shapes can be encoded and amplified in tandem with controllable release of specific shapes from the amplicons, thereby providing a convenient yet economical way to safely store “genetic codes” of nanostructures on an ssDNA for selective mass production on demand. We expect our approach will promote the development of applications of DNA catalysts as well as gene-sized ssDNA in broad areas.

A high density CHO-S transient transfection system: Comparison of ExpiCHO and Expi293

Chinese Hamster Ovary (CHO) cells are the principal mammalian host used for stable cell line generation and biotherapeutic protein production. Until recently, production of milligrams to grams of protein in CHO transient systems was challenging. As such, Human Embryonic Kidney (HEK293) cells are the most common mammalian cell type used for transient transfection. The post-translational modifications (PTMs) of a protein are dictated in part by the cell line used for expression, and changes in PTMs have been shown to affect both the activity and biophysical properties of proteins. Therefore, it is potentially advantageous to keep the host cell type consistent throughout drug discovery and development. To this end, we compared the ExpiCHO system, a high density CHO-S transient transfection system, to the Expi293 and FreeStyle MAX CHO transient systems. Fourteen proteins were expressed in both the Expi293 and ExpiCHO systems. For a majority of proteins tested, the protein titers observed with the ExpiCHO system were higher than those seen with both the FreeStyle MAX CHO and Expi293 systems. Antibodies expressed using the ExpiCHO system had glycosylation patterns more similar to antibodies produced in stable CHO cell lines than Expi293-derived antibodies. However, culture duration and temperature were found to affect protein titer, monodispersity, enzyme activity, and PTMs and should be carefully selected when using the ExpiCHO system. The ExpiCHO transient transfection systems allows for facile production of milligrams to grams of protein in CHO cells and de-risks the transition from transient to stable material during drug development.

Production of IgGs with a human-like sialylation in CHO cells

Background IgGs that possess a2,6-sialylated Fc-glycans are thought to be responsible for the anti-inflammatory properties of intravenous immunoglobulins (IVIGs) [1], through a mechanism which has not been elucidated yet. The impact of this sialylation on the classic IgG’s effector functions also remains unclear [2,3]. The understanding of these mechanisms has been impeded by the heterogeneity of the sialylated glycan species together with the relative rarity of a2,6-sialylated IgGs. N-glycan sialylation is a post-translational modification where a sialic acid (SA) residue is added to the terminal galactose residue (Gal) of a growing glycan chain. The SA type (NANA or NGNA), the nature of the linkage between the SA and the Gal (a2,3 or a2,6), or the number of glycan antennae being sialylated, may vary according to the IgG subtype, the host cell in which it is expressed and the cell culture environment. A closer attention must thus be paid to the precise sialylation profiles leading to the reduced ADCC activity and to the gain of anti-inflammatory properties. In humans, 10 to 15% of the circulating IgG1s are sialylated, carrying mostly complex di-antennary glycans with two Gal and one a2,6-linked SA residue (G2FS(6)1, where G stands for galactose, F for fucose and S(6) for a2,6SA). Most of the therapeutic monoclonal antibodies (mabs) are produced in Chinese hamster ovary (CHO) cells, which have a glycosylation machinery close to that of humans, but possess only a2,3-sialyltransferases (ST3) whereas humans have both a2,3and a2,6-sialyltransferases. The Fc domain of mabs produced in CHO typically possesses N-glycans with low galactosylation and very low sialylation (0-2% of a2,3-sialylated glycans). In this study, we show that the a2,6-sialylation of IgG1’s Fc domain can be efficiently achieved by the transient coexpression of the human b1,4-galactosyltransferase 1 (GT) and a2,6-sialyltransferase 1 (ST6) in CHO cells, whereas the expression of one or the other glycosyltransferase alone does not significantly improve sialylation [4]. The process allows for the production of milligrams of human-like sialylated mabs within two weeks. We present a panel of four orthogonal assays for the fine characterization of the mabs’ glycoprofile that are in very good agreement with each other.

Recombinant production of human ICAM-1 chimeras by single step on column refolding and purification

The interaction of the adhesion molecule of the immunoglobulin family intercellular adhesion molecule 1 (ICAM-1) with its ligands such that the integrins LFA-1 and Mac-1 is crucial for the regulation of several physiological and pathophysiological processes like cell mediated-elimination of tumor or virus infected cells, cancer metastasis or inflammatory autoimmune processes. Thus, production of milligrams of protein is required to perform structural and functional studies as well as design novel approaches to find out new inhibitors of ICAM-1/LFA-1 interaction. Here we report on the production of a recombinant human ICAM-1 chimera comprising the first two extracellular domains of ICAM-1 linked to the Fc fraction of a human IgG1. To this aim we have used a cost-effective method based on the expression of a His-tagged protein in Escherichia coli followed by a single step of refolding and purification on immobilized metal affinity columns. This method is able to produce 3mg/l of bacterial culture in just 72h with purity greater than 95%. The identity and the native structure of refolded human ICAM-1 chimera were confirmed by biochemical and biophysical studies including SDS-electrophoresis, immunoblot, circular dichroism, isothermal titration calorimetry and fluorescence spectroscopy. Native folding and functional activity of the chimera were further confirmed by different cell biology studies, including B cell adhesion, T cell binding and inhibition of NK cell function. These studies indicate a high biological activity of the protein since it induces a 200-fold increase/mg of protein in B cell adhesion and the inhibitory dose 50 to block cell-mediated cytotoxicity is 10pg/effector cell. These analyses show that our protocol is able to produce a recombinant human ICAM-1 chimera fully active and useful to analyze the biological processes in which ICAM-1/LFA-1 interaction is critically involved.

Enzymatic production of mono-ubiquitinated proteins for structural studies: The example of the Josephin domain of ataxin-3

Protein ubiquitination occurs through formation of an isopeptide bond between the C-terminal glycine of ubiquitin (Ub) and the ɛ-amino group of a substrate lysine residue. This post-translational modification, which occurs through the attachment of single and/or multiple copies of mono-ubiquitin and poly-ubiquitin chains, is involved in crucial cellular events such as protein degradation, cell-cycle regulation and DNA repair. The abnormal functioning of ubiquitin pathways is also implicated in the pathogenesis of several human diseases ranging from cancer to neurodegeneration. However, despite the undoubted biological importance, understanding the molecular basis of how ubiquitination regulates different pathways has up to now been strongly limited by the difficulty of producing the amounts of highly homogeneous samples that are needed for a structural characterization by X-ray crystallography and/or NMR. Here, we report on the production of milligrams of highly pure Josephin mono-ubiquitinated on lysine 117 through large scale in vitro enzymatic ubiquitination. Josephin is the catalytic domain of ataxin-3, a protein responsible for spinocerebellar ataxia type 3. Ataxin-3 is the first deubiquitinating enzyme (DUB) reported to be activated by mono-ubiquitination. We demonstrate that the samples produced with the described method are correctly folded and suitable for structural studies. The protocol allows facile selective labelling of the components. Our results provide an important proof-of-concept that may pave the way to new approaches to the in vitro study of ubiquitinated proteins.

Culture of HEK293-EBNA1 Cells for Production of Recombinant Proteins

INTRODUCTIONFast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Pure r-proteins are often required in large amounts (hundreds of milligrams to gram quantities) when being developed as biotherapeutics, or in smaller quantities (milligrams) for high-throughput screening campaigns and structural or functional studies. Mammalian cells are often preferred over prokaryotic systems when expressing cDNAs of mammalian origin due to their superior capability to conduct elaborate post-translational modifications. Large-scale transfection of mammalian cells is now establishing itself as a "must-have" technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins within a few days after cDNA cloning into the appropriate expression vector. The HEK293 cell line stably expressing the Epstein-Barr virus nuclear antigen-1 (HEK293-EBNA1, or 293E) is the most commonly used cell line for large-scale transfection. When using expression vectors bearing the Epstein-Barr virus origin of replication, oriP (such as the pTT vector), a threefold improvement in r-protein yield is generally obtained over a similar non-oriP vector. This protocol describes a method for culturing HEK293-EBNA1 cells which will then be used to produce recombinant proteins.

Transfection of HEK293-EBNA1 Cells in Suspension with Linear PEI for Production of Recombinant Proteins

INTRODUCTIONFast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Pure r-proteins are often required in large amounts (hundreds of milligrams to gram quantities) when being developed as biotherapeutics, or in smaller quantities (milligrams) for high-throughput screening campaigns and structural or functional studies. Mammalian cells are often preferred over prokaryotic systems when expressing cDNAs of mammalian origin due to their superior capability to conduct elaborate post-translational modifications. Large-scale transfection of mammalian cells is now establishing itself as a "must-have" technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins within a few days after cDNA cloning into the appropriate expression vector. Although calcium-mediated large-scale transfection is very effective, it is usually achieved in serum-containing medium under tightly controlled conditions that are difficult to achieve on a large scale. In contrast, polyethylenimine (PEI) is much easier to use: It binds to and precipitates DNA efficiently and the resulting DNA-PEI complexes are suitable for efficient transfection of mammalian cells. PEI has been used successfully on a large scale in serum-containing and serum-free cultures. In particular, the linear isoform of PEI is more effective for transfecting cells in suspension. This protocol describes the steps needed for successful transfection of HEK293 cells adapted to serum-supplemented or serum-free medium in suspension culture using linear PEI.

Transfection of Adherent HEK293-EBNA1 Cells in a Six-Well Plate with Branched PEI for Production of Recombinant Proteins

INTRODUCTIONFast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Pure r-proteins are often required in large amounts (hundreds of milligrams to gram quantities) when being developed as biotherapeutics, or in smaller quantities (milligrams) for high-throughput screening campaigns and structural or functional studies. Mammalian cells are often preferred over prokaryotic systems when expressing cDNAs of mammalian origin due to their superior capability to conduct elaborate post-translational modifications. Large-scale transfection of mammalian cells is now establishing itself as a "must-have" technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins within a few days after cDNA cloning into the appropriate expression vector. Although calcium-mediated large-scale transfection is very effective, polyethylenimine (PEI) is much easier to use: It binds to and precipitates DNA efficiently and the resulting DNA-PEI complexes are suitable for efficient transfection of mammalian cells. In particular, the branched isoform of PEI works well for adherent cells, as it promotes their attachment to the plastic surface. It is thus very useful in experiments requiring multiple medium exchanges or washing steps following transfection. Also, when used in conjunction with six-well CellBIND plates, branched PEI can be used to adhere transfected cells when establishing stable cell lines. This protocol describes the steps needed for successful transfection of HEK293 cells adapted to serum-supplemented or serum-free medium in adherent culture using branched PEI.

Transfection of HEK293-EBNA1 Cells in Suspension with 293fectin for Production of Recombinant Proteins

INTRODUCTIONFast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Pure r-proteins are often required in large amounts (hundreds of milligrams to gram quantities) when being developed as biotherapeutics, or in smaller quantities (milligrams) for high-throughput screening campaigns and structural or functional studies. Mammalian cells are often preferred over prokaryotic systems when expressing cDNAs of mammalian origin, due to their superior capability to conduct elaborate post-translational modifications. Large-scale transfection of mammalian cells is now establishing itself as a "must-have" technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins within a few days after cDNA cloning into the appropriate expression vector. Although calcium-mediated large-scale transfection is very effective, other methods suitable for efficient transfection of mammalian cells are easier to use. This protocol describes the steps needed for successful transfection of 293-6E cells in suspension culture in serum-free medium using 293fectin.

Transient gene expression in HEK293 cells: Peptone addition posttransfection improves recombinant protein synthesis

Gene expression by large-scale transfection of mammalian cells is becoming an established technology for the fast production of milligram and even gram amounts of recombinant proteins (r-proteins). However, efforts are still needed to optimize production parameters in order to maximize volumetric productivities while maintaining product quality. In this study, transfection efficiency and volumetric productivity following transient gene expression in HEK293 cells were evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphatase (SEAP) as reporter genes. We show that a single pulse of peptones (protein hydrolysates) to the cultures performed in a low serum (1%, v/v) and in serum-free medium results in a significant increase in volumetric protein productivity. Sixteen peptones from different sources were tested and almost all of them showed a positive effect on r-protein production. This effect, however, is time- and concentration-dependent. By using Tryptone N1 (a casein peptone, TN1) to feed the cultures at 24 h posttransfection (hpt), a 2-fold increase in volumetric SEAP productivity was obtained 5 days posttransfection. This effect was shown to be equal to that obtained when the culture was fed with a supplementary 4% (v/v) of serum. The positive effect of TN1 on protein production was also demonstrated with Tie2 protein ectodomain produced in serum-free medium. HPLC analysis of amino acids consumption/production during control batch and TN1 pulse culture showed some major differences in amino acid metabolism when using TN1 pulse. Asparagine, glycine, histidine, threonine, leucine, and valine show accumulation in the medium over the cultivation period instead of being consumed as observed in unfed sample (except for asparagine, which remained unchanged). Isoleucine, tyrosine, methionine, and phenylalanine all remained unchanged or slightly fluctuated in TN1-fed culture after the feeding pulse, while they were all steadily consumed in the control run. The relative abundance of SEAP's mRNA suggests that the improvement in protein yield results both from an increase of the translational activity and transcription efficiency. Further understanding of mechanisms by which amino acids/peptides regulate transcriptional and translational machinery in mammalian cells should facilitate the design of new strategies for the improvement of r-protein production by large-scale transfection.

Production of milligram concentrations of free prostate specific antigen (fPSA) from LNCaP cell culture: Difference between fPSA from LNCaP cell and seminal plasma

We have established a procedure for the production of milligrams of free PSA (fPSA) from LNCaP cells derived from a human carcinoma of the prostate. By growing LNCaP cells in a serum-free medium in the presence of a synthetic androgen (R1881) and taking advantage of the special design of the Micro-mouse Hollow Fiber Bioreactor, relatively pure fPSA could be obtained. We found that columns containing either Sephacryl S-100 or S-200 could be used to remove the small amount of bovine serum albumin (BSA) and PSA-α1-antichymotrypsin complex (PSA-ACT) from the preparation. More than 90% of the PSA from LNCaP cell cultures are fPSA. Like fPSA from seminal plasma, two fractions of fPSA differing in protease activity can be separated by DEAE-Sepharose chromatography. Based on the band pattern exhibited on the Western blot following sodium dodecyl sulfate-polyacrylamide electrophoresis separation, fPSA from LNCaP contains more inactive PSA isoforms. This was confirmed by chromatofocusing: the isoelectric point (pI) of the major PSA isoforms from the LNCaP cell culture were higher (6.8 and 6.6) than that (6.4 and 6.1) of fPSA from seminal fluid. We conclude that the LNCaP cell culture is a reliable source for obtaining large quantities of pure fPSA both for the preparation of assay calibrators and controls and for studying the difference in fPSA between benign prostate disease and prostate cancer. J. Clin. Lab. Anal. 12:6–13, 1998. © 1998. Wiley-Liss, Inc.

Production of milligram concentrations of free prostate specific antigen (fPSA) from LNCaP cell culture: Difference between fPSA from LNCaP cell and seminal plasma

We have established a procedure for the production of milligrams of free PSA (fPSA) from LNCaP cells derived from a human carcinoma of the prostate. By growing LNCaP cells in a serum-free medium in the presence of a synthetic androgen (R1881) and taking advantage of the special design of the Micro-mouse Hollow Fiber Bioreactor, relatively pure fPSA could be obtained. We found that columns containing either Sephacryl S-100 or S-200 could be used to remove the small amount of bovine serum albumin (BSA) and PSA-alpha 1-antichymotrypsin complex (PSA-ACT) from the preparation. More than 90% of the PSA from LNCaP cell cultures are fPSA. Like fPSA from seminal plasma, two fractions of fPSA differing in protease activity can be separated by DEAE-Sepharose chromatography. Based on the band pattern exhibited on the Western blot following sodium dodecyl sulfate-polyacrylamide electrophoresis separation, fPSA from LNCaP contains more inactive PSA isoforms. This was confirmed by chromatofocusing: the isoelectric point (pl) of the major PSA isoforms from the LNCaP cell culture were higher (6.8 and 6.6) than that (6.4 and 6.1) of fPSA from seminal fluid. We conclude that the LNCaP cell culture is a reliable source for obtaining large quantities of pure fPSA both for the preparation of assay calibrators and controls and for studying the difference in fPSA between benign prostate disease and prostate cancer.