Single step protocol to purify recombinant proteins with low endotoxin contents

Endotoxin is an unwanted by product of recombinant proteins purified from Escherichia coli. The inherent toxicity of endotoxins makes their removal an important step for the proteins' application in several biological assays and for safe parenteral administration. The method described in this paper is a one-step protocol which is effective at removing tightly bound endotoxin from over-expressed tagged proteins in E. coli. We combined affinity chromatography with a non-ionic detergent washing step, to remove most of the endotoxin contaminants from the end product. An endotoxin reduction of less than 4 to 0.2 EU mg(-1) was achieved with protein recovery close to a yield 100%. As this new protocol requires only one step to simultaneously purify tagged proteins and eliminate endotoxins, it represents a substantial advantage in time, effort, and expense.     

A peptide carrier for the delivery of biologically active proteins into mammalian cells.

The development of peptide drugs and therapeutic proteins is limited by the poor permeability and the selectivity of the cell membrane. There is a growing effort to circumvent these problems by designing strategies to deliver full-length proteins into a large number of cells. A series of small protein domains, termed protein transduction domains (PTDs), have been shown to cross biological membranes efficiently and independently of transporters or specific receptors, and to promote the delivery of peptides and proteins into cells. TAT protein from human immunodeficiency virus (HIV-1) is able to deliver biologically active proteins in vivo and has been shown to be of considerable interest for protein therapeutics. Similarly, the third alpha-helix of Antennapedia homeodomain, and VP22 protein from herpes simplex virus promote the delivery of covalently linked peptides or proteins into cells. However, these PTD vectors display a certain number of limitations in that they all require crosslinking to the target peptide or protein. Moreover, protein transduction using PTD-TAT fusion protein systems may require denaturation of the protein before delivery to increase the accessibility of the TAT-PTD domain. This requirement introduces an additional delay between the time of delivery and intracellular activation of the protein. In this report, we propose a new strategy for protein delivery based on a short amphipathic peptide carrier, Pep-1. This peptide carrier is able to efficiently deliver a variety of peptides and proteins into several cell lines in a fully biologically active form, without the need for prior chemical covalent coupling or denaturation steps. In addition, this peptide carrier presents several advantages for protein therapy, including stability in physiological buffer, lack of toxicity, and lack of sensitivity to serum. Pep-1 technology should be extremely useful for targeting specific protein-protein interactions in living cells and for screening novel therapeutic proteins.     

Highly efficient selenomethionine labeling of recombinant proteins produced in mammalian cells

The advent of the multiwavelength anomalous diffraction phasing method has significantly accelerated crystal structure determination and has become the norm in protein crystallography. This method allows researchers to take advantage of the anomalous signal from diverse atoms, but the dominant method for derivative preparation is selenomethionine substitution. Several generally applicable, high-efficiency labeling protocols have been developed for use in the bacterial, yeast, and baculovirus/insect cell expression systems but not for mammalian tissue culture. As a large number of proteins of biomedical importance can only be produced in yields sufficient for X-ray diffraction experiments in mammalian expression systems, it becomes all the more important to develop such protocols. We therefore evaluated several variables that play roles in determining incorporation levels and report here a simple protocol for selenomethionine modification of proteins in mammalian cells routinely yielding >90% labeling efficiency.     

Novel approach to obtain biologically active recombinant heterodimeric proteins in Escherichia coli

The strategy described in this paper provides a novel approach for recombinant expression of heterodimeric proteins, and is especially suitable for the production of proteins whose characteristics lead to aggregation in E. coli expression systems. Pheromaxein, a steroid-binding protein isolated from boar saliva, is a heterodimeric protein consisting of 10x10(3) rel. mol. mass units (pheromaxein A) and 8x10(3) rel. mol. mass units (pheromaxein C) subunits. Expression of pheromaxein subunits in E. coli resulted in extensive insoluble aggregation. The difficulty faced in obtaining soluble recombinant pheromaxein subunits was clearly evident when native pheromaxein immediately formed aggregates when it was separated into its individual subunits. An increase in soluble pheromaxein expression in E. coli was obtained when the subunits were expressed as fusion proteins with thioredoxin. Pheromaxein genes were inserted separately into pET32a+ vectors at the NcoI site, resulting in thioredoxin, S.Tag and His.Tag coding regions being upstream of the inserted gene. Soluble pheromaxein A-thioredoxin (pheroA/trx) and pheromaxein C-thioredoxin (pheroC/trx) fusions were purified to homogeneity, using a laboratory scale S-protein agarose affinity column. Cleavage of thioredoxin under normal conditions was not feasible due to the extensive aggregation problems experienced when pheromaxein subunits exist separately. PheroA/trx and pheroC/trx were therefore mixed together and cleaved from thioredoxin simultaneously so that pheromaxein subunits were given an instant opportunity to associate under oxido-shuffling conditions. The glutathione oxido-shuffling system allowed the disulphide bridges between pheromaxein A and C to rearrange until the correct native structure was formed. This novel approach combines affinity purification with a coupled fusion protein-cleavage and refolding technique.     

High-level production and purification of biologically active proteins from bacterial and mammalian cells using the tandem pGFLEX expression system

Because of the complexities involved in the regulation of gene expression in Escherichia coli and mammalian cells, it is considered general practice to use different vectors for heterologous expression of recombinant proteins in these host systems. However, we have developed and report a shuttle vector system, pGFLEX, that provides high-level expression of recombinant glutathione S-transferase (GST) fusion proteins in E. coli and mammalian cells. pGFLEX contains the cytomegaloma virus (CMV) immediate-early promoter in tandem with the E. coli lacZpo system. The sequences involved in gene expression have been appropriately modified to enable high-level production of fusion proteins in either cell type. The pGFLEX expression system allows production of target proteins fused to either the N or C terminus of the GST π protein and provides rapid purification of target proteins as either GST fusions or native proteins after cleavage with thrombin. The utility of this vector in identifying and purifying a component of a multi-protein complex is demonstrated with cyclin A. The pGFLEX expression system provides a singular and widely applicable tool for laboratory or industrial production of biologically active recombinant proteins in E. coli and mammalian cells.     

A strategy for the expression of recombinant proteins traditionally hard to purify

We have developed a series of plasmid vectors for the soluble expression and subsequent purification of recombinant proteins that have historically proven to be extremely difficult to purify from Escherichia coli. Instead of dramatically overproducing the target protein, it is expressed at a low basal level that facilitates the correct folding of the recombinant protein and increases its solubility. Highly active recombinant proteins that are traditionally difficult to purify are readily purified using standard affinity tags and conventional chromatography. To demonstrate the utility of these vectors, we have expressed and purified full-length human DNA polymerases η, ι, and ν from E. coli and show that the purified DNA polymerases are catalytically active in vitro.     

Simple immunoaffinity method to purify recombinant hepatitis B surface antigen secreted by transfected mammalian cells

Purification of recombinant hepatitis B surface antigen (recHBsAg) produced in a stable Chinese hamster ovary (CHO) cell line was evaluated using Linx Affinity Purification System (Invitrogen, USA). To purify HBsAg secreted by this cell line, a murine monoclonal antibody (MAbAH1) raised against native HBsAg was used. The purified AH1MAb was conjugated with phenyldiboronic acid (PDBA) and immobilized on the immunoaffinity chromatographic support. Using an optimized protocol the affinity column was able to purify recHBsAg from supernatant of mammalian cells cultures with more than 80% purity. This method showed to be simple and quicker than the current ultracentrifugation methods. The method is also efficient and economical in obtaining purified recHBsAg.     

Design of transgenes for efficient expression of active chimeric proteins on mammalian cells

Heterologous proteins expressed on the surface of cells may be useful for eliciting therapeutic responses and engineering new extracellular properties. We examined factors that control the membrane targeting of alpha-fetoprotein (AFP) and a single-chain antibody (scFv). Chimeric proteins were targeted to the plasma membrane by employing the transmembrane domain (TM) and cytosolic tail of murine CD8O (B7-1), the TM of the human platelet-derived growth factor receptor (PDGFR), the glycosylphosphatidylinositol anchor encoded by the C-terminal extension of decay-accelerating factor (DAF), and the TM of the H1 subunit of the human asialoglycoprotein receptor (ASGPR). AFP chimeric proteins containing the B7, DAF, ASGPR, or PDGFR targeting domains displayed half-lives of 12.2, 3.8, 2.4, and 1.6 h, respectively. The newly synthesized B7 chimera was rapidly transported and remained on the cell surface. Glycosylphosphatidylinositol-anchored chimeras reached the surface more slowly and significant amounts were released into the culture medium. PDGFR TM chimeras were rapidly degraded, whereas ASGPR chimeras were retained in the endoplasmic reticulum (ER). The surface expression of both AFP and scFv chimeric proteins followed the order (highest to lowest) of B7 > DAF > PDGFR. Introduction of a dimerization domain (hinge-CH(2)-CH(3) region of human IgG1) between scFv and TM dramatically reduced cleavage of the chimeric protein, increased surface expression, and produced biologically active scFv. Our results indicate that transgenes designed for the expression of active scFv on cells should incorporate a TM that does not undergo endocytosis, include an intact cytoplasmic domain, and possess a spacer to reduce cleavage and retain biological activity.     

Engineering a noncarrier to a highly efficient carrier peptide for noncovalently delivering biologically active proteins into human cells

Noncovalent protein delivery into cells via peptide carriers is an emerging concept. Only a handful of such peptides are known. To address various limitations associated with protein delivery for therapeutic purposes, a greater number of different delivery peptides would be required. No general method exists for creating such peptides. By combining a sequence of 16 lysine residues (K16) with the signal peptide (SP) sequence of Kaposi's fibroblast growth factor (K-FGF), we have synthesized a peptide (K16SP) that efficiently and noncovalently delivers functionally intact proteins (immunoglobulin G molecules, beta-galactosidase, and green fluorescent protein) into mammalian cells. The peptides K16 and SP each alone did not show any noncovalent protein-carrying capacity. K16SP appears to be nontoxic to cells and three to four times more efficient than a commercially available peptide reagent. Our approach offers proof-of-concept of a general strategy for creating a diverse array of peptide carriers for eventual therapeutic applications.     

Versatile method for production and controlled polymer-immobilization of biologically active recombinant proteins

The immobilization of a protein by covalent attachment to a support matrix should involve only functional groups of the protein that are not essential for its biological activity. A general strategy for obtaining recombinant proteins designed for oriented covalent grafting onto copolymers was investigated. The rationale involves the definition of seven p24-derived recombinant proteins as fused to either distant or adjacent tags comprising primary amine rich tag consisting of six contiguous lysines suitable for oriented covalent immobilization and a hexa-histidine tag suitable for metal chelate affinity purification. High-level expression, efficient affinity purification, and coupling yields onto maleic anhydride-alt-methyl vinyl ether copolymers higher than 95% were obtained for all proteins. Afterwards, an investigation of the biological features of the immobilized vs. nonimmobilized protein onto the copolymer allowed us to select one bioconjugate which was used in a diagnostic context, i.e., as a capture antigen in an ELISA format test. Sera from 107 HIV-seropositive individuals at various stages of HIV infection, including two seroconversion panels and 104 healthy HIV-seronegative controls, were tested using either RH24 or RK24H-copolymer coated onto the microtiter plate. These assays showed that the use of such a protein-copolymer bioconjugate allowed detection of lower antibody titers than the RH24 protein, illustrating the potential of applications of such doubly tagged proteins. Thus, a set of expression vectors was designed containing four different combinations of hexa-lysine and hexa-histidine tags and a multiple cloning site, allowing the production of different recombinant fusion proteins suitable for biological reactivity conservation after immobilization.     

High-level expression of biologically active human prolactin from recombinant baculovirus in insect cells

We examined the feasibility of high-level production of recombinant human prolactin, a multifunctional protein hormone, in insect cells using a baculovirus expression system. The human prolactin cDNA with and without the secretory signal sequence was cloned into pFastBac1 baculovirus vector under the control of polyhedrin promoter. Prolactin was produced upon infection of either Sf9 or High-Five cells with the recombinant baculovirus containing the human prolactin cDNA. The production of recombinant prolactin varied from 20 to 40 mg/L of monolayer culture, depending on the cell types. The prolactin polypeptide with its own secretory signal was secreted into the medium. N-terminal amino acid sequence analysis of the recombinant polypeptide purified from the culture medium indicated that the protein was processed similar to human pituitary prolactin. Carbohydrate analysis of the purified protein indicated that a fraction of the recombinant prolactin made in insect cells appeared to be glycosylated. Also, both secreted and nonsecreted forms of the recombinant prolactin in insect cells were biologically equivalent to the native human prolactin (pituitary derived) in the Nb2 lymphoma cell proliferation assay.     

A simple, rapid and large capacity ELISA for biologically active native and recombinant human IFN gamma

A rapid and sensitive enzyme-immunoassay for native and recombinant human interferon gamma is described. The test is performed in one step at room temperature and is based on the sandwich principle. The IFN gamma preparation is distributed with horse radish peroxidase-labeled monoclonal antibody to IFN gamma in microtiter plates previously coated with a second mab against IFN gamma. The amount of the IFN gamma mab sandwich fixed in the microtiter plate wells is proportional to the color developed after the addition of peroxidase-specific substrate. The two mab's used in the test neutralize IFN gamma and are directed against the same epitope. For this reason they can only detect the biologically active dimeric form of IFN gamma. The IFN gamma-ELISA works in phosphate buffer as well as in tissue culture medium or human serum. As the assay is routinely performed in 2 hours, the limit of detection is 3 U/ml of IFN gamma (0.3 ng/ml). If the assay is performed in 16 hours, the limit of detection decreases to 0.5 U/ml IFN gamma (0.06 ng/ml). The conditions to preserve the activity of IFN gamma preparation as standard are discussed.     

Expression and purification of active recombinant ATM protein from transiently transfected mammalian cells

The gene mutated in the human disease ataxia telangiectasia (AT), termed ATM, encodes a large protein kinase involved in DNA repair and cell cycle control. Biochemical characterization of ATM function has been somewhat difficult because of its large size (approximately 370 kDa) and relatively low level of expression in several systems. The majority of studies have used immunoprecipitated ATM or purified ATM obtained through relatively complex procedures. Here, we describe an efficient method for the expression and purification of FLAG-epitope-tagged recombinant human ATM protein (F-ATM). This method utilizes the expression of F-ATM in transiently transfected 293T cells followed by anti-FLAG-agarose affinity chromatography. The transfection procedure has been optimized for large (225-cm(2)) culture flasks and F-ATM can be purified to near homogeneity as judged by SDS-PAGE. This procedure yields approximately 1 microg of catalytically active F-ATM protein/225-cm(2) flask that can be used for biochemical studies.     

Expression of biologically active rat prolactin in mammalian COS-1 cells

Rat prolactin (PRL) cDNA was constructed in mammalian expression vector, pSVL. Transient expression of rat PRL was performed in COS-1 cells by the DEAE-dextran method. The production of recombinant rat PRL started within 48 h from the cells and reached the level of 1.0-1.5 micrograms/ml/5 x 10(5) cells. The molecular size of recombinant rat PRL was the same as that of standard rat PRL (Mw: 23,000), suggesting successful removal of the signal peptide. The radioimmunoassay and isoelectric focusing analysis showed that recombinant rat PRL has almost the same immunological and biochemical characteristics as those of standard rat PRL. As biological tests, receptor-binding activity, Nb 2 node lymphoma cell growth activity, and mammary gland stimulating activity were examined. The radioreceptor assay showed that recombinant rat PRL has binding activity to mammary microsomal membrane similar to that of standard rat PRL. Recombinant rat PRL also stimulated the growth of Nb 2 lymphoma cells as standard rat PRL. Finally it was shown that recombinant rat PRL promotes the synthesis of the secretory materials in the lumen of mouse mammary gland with the same potency as that of standard rat PRL. In conclusion, recombinant rat PRL, which was produced in mammalian cells in the present experiment, has immunological, biochemical and biological characteristics similar to those of standard PRL, and has full bioactivity.     

Precise and efficient cleavage of recombinant fusion proteins using mammalian aspartic proteases

Expression of recombinant proteins as translational fusions is commonly employed to enhance stability, increase solubility and facilitate purification of the desired protein. In general, such fusion proteins must be cleaved to release the mature protein in its native form. The usefulness of the procedure depends on the efficiency and precision of cleavage and its cost per unit activity. We report here the development of a general procedure for precise and highly efficient cleavage of recombinant fusion proteins using the protease chymosin. DNA encoding a modified pro-peptide from bovine chymosin was fused upstream of hirudin, carp growth hormone, thioredoxin and cystatin coding sequences and expressed in a bacterial Escherichia coli host. Each of the resulting fusion proteins was efficiently cleaved at the junction between the pro-peptide and the desired protein by the addition of chymosin, as determined by activity, N-terminal sequencing and mass spectrometry of the recovered protein. The system was tested further by cleavage of two fusion proteins, cystatin and thioredoxin, sequestered on oilbody particles obtained from transgenic Arabidopsis seeds. Even when the fusion protein was sequestered and immobilized on oilbodies, precise and efficient cleavage was obtained. The precision, efficiency and low cost of this procedure suggest that it could be used in larger scale manufacturing of recombinant proteins which benefit from expression as fusions in their host organism.     

A novel strategy to over-express and purify homologous proteins from Streptococcus pneumoniae

Functional studies of Streptococcus pneumoniae virulence factors are facilitated by the development of complementation/mutagenesis systems. These methods usually result in poor expression yields; therefore, biochemical and structural/functional characterizations are mostly performed with proteins expressed and purified from heterologous systems (e.g. Escherichia coli). However, heterologous expression does not guarantee correct protein structure and function. In this work, we developed a method to over-express and purify homologous proteins from S. pneumoniae. The system relies on the combined use of the shuttle plasmid pMU1328 and a natural constitutive pneumococcal promoter, P₉₆. Efficient over-expression of secreted, membrane or surface anchored proteins, either wild type or mutant, was achieved. As proof of principle the S. pneumoniae pilus-1 backbone RrgB was successfully purified as a His-tag secreted protein (RrgB-His_SP) from pneumococcal culture supernatants. N-terminal sequencing and mass spectrometry analysis of RrgB-His_SP allowed the determination of the leader sequence cleavage site in pneumococcus, while proteolysis studies confirmed the stability of RrgB-His_SP to trypsin digestion. The data presented here support the use of this novel homologous expression method for all S. pneumoniae proteins for which extensive characterization studies are planned. Moreover, given the promiscuity of the pMU1328 replicon, this system could be used in diverse bacterial species.     

Intranuclear uptake and persistence of biologically active DNA after electroporation of mammalian cells

Radiolabeled or biologically functional DNA molecules were introduced into cells by electroporation in a variety of forms: double stranded circles, linearized double stranded fragments and single stranded circular molecules. Molecules rapidly entered cells after exposure to a high field-strength electric pulse and then redistributed between the cytoplasm and the nucleus. Maximal intranuclear levels approximated 10(4) molecules per cell. Introduced DNA persisted in a biologically active form with a half-life of 15-24 h. There was no evidence for biologically significant alteration of two double stranded gene sequences. Single stranded DNA molecules also retained biological activity.     

The expression of biologically active human p53 in Leishmania cells: a novel eukaryotic system to produce recombinant proteins.

We have investigated the use of Leishmania cells as a novel eukaryotic expression system for the production of recombinant protein. These cells are easy to maintain, requiring no CO2 incubator or shaker, and can be grown in standard tissue culture media. Leishmania cells can be readily transfected with plasmid DNA by electroporation and transformants selected with antibiotic resistance. Recent studies have shown that it is possible to express foreign genes in Leishmania for the purpose of understanding the biology of this protozoan cell. In the present study we report the use of this system as a means of producing a biologically functional human p53 protein. The conformation of the p53 protein is critical for its ability to bind specific DNA sequences. It is demonstrated that Leishmania-synthesized human p53 is phosphorylated and can bind specifically to its enhancer DNA sequence. These data demonstrate that Leishmania may represent a simple eukaryotic expression system for the production of biologically active recombinant proteins.