Eukaryotic integral membrane protein expression utilizing the <Emphasis Type="Italic">Escherichia coli</Emphasis> glycerol-conducting channel protein (GlpF)

A fusion protein expression system is described that allows for production of eukaryotic integral membrane proteins in Escherichia coli (E. coli). The eukaryotic membrane protein targets are fused to the C terminus of the highly expressed E. coli inner membrane protein, GlpF (the glycerol-conducting channel protein). The generic utility of this system for heterologous membrane-protein expression is demonstrated by the expression and insertion into the E. coli cell membrane of the human membrane proteins: occludin, claudin 4, duodenal ferric reductase and a J-type inwardly rectifying potassium channel. The proteins are produced with C-terminal hexahistidine tags (to permit purification of the expressed fusion proteins using immobilized metal affinity chromatography) and a peptidase cleavage site (to allow recovery of the unfused eukaryotic protein).     

Expression of selected domains of the circumsporozoite antigen ofPlasmodium knowlesi

The circumsporozoite antigen of the simian malarial parasite,Plqsmodium knowlesi, consists of tandemly repeated immunodominant peptide units which may play a role in evading the immune system. To study the immunogenicity of this antigen in the absence of the immunodominant repeats, the whole of the non-repetitive region of this antigen has been expressed inEscherichia coli. The entire amino-terminal region up to the start of the repeats, and the full non-repetitive carboxyl region starting from the end of the repeats up to the termination codon, have been expressed separately, as fusion proteins with a 26 kD glutathione-S-transferase protein ofSchistosomq japonicum. A repeat-less truncated antigen has also been expressed as the same fusion protein. The amino-terminal fusion protein (GST-CSN), is a soluble protein of a molecular weight of 38 kD, which could be purified by affinity chromatography on immobilized glutathione. The carboxylterminal fusion protein (GST-CSC), is insoluble, migrates with an anomalous molecular weight of 32 kD, and binds to the affinity matrix weakly. The truncated repeat-less fusion protein (GST-CSNC) is also, an insoluble protein of molecular weight of 48 kD. Unlike the two separate domains, GST-CSNC is an extremely unstable protein inEscherichia coli.     

Recombinant proteins L and LG

Several bacterial cell wall proteins, like proteins A and G, with valuable affinity for immunoglobulins have been discovered and are currently employed in immunological techniques. In 1988, protein L, a bacterial cell wall protein with Ig-binding capacity, was isolated from the anaerobic bacterial speciesPeptostreptococcus magnus. Binding data with immunoglobulin fragments suggested that protein L could selectively bind the variable region of human kappa light chains. More recently a recombinant LG fusion protein was expressed inE. coli containing four repeated Ig-binding domains of protein L (fragment B1–4) and two IgG Fc-binding protein G domains (fragment CDC). Recombinant L and LG proteins were tested in the purification of murine monoclonal IgG and their fragments. After affinity-constant evaluation in different buffer systems, high-pressure affinity-chromatography columns were prepared by coupling the proteins to Affi-prep 10 resin and tested with eight different murine monoclonal antibodies and their fragments of various isotypes. Affinity-chromatography experiments confirming radioimmunoassay results showed 75% fragment-binding capacity of protein L and 100% of protein LG. These results evidenced protein LG as the most powerful Ig-binding tool so far described. Therefore, application of these proteins may be suggested in the purification of murine immunoglobulins and their fragments, including the engineered ones.     

Plasmid pGEX-5T: An alternative system for expression and purification of recombinant proteins

Summary A novel expression vector pGEX-5T was constructed which directs the synthesis of a fusion protein with a histidine-hexapeptide and glutathione-S-transferase at its N-terminus and the recombinant protein at its C-terminus inEscherichia coli. The designed fusion gene strategy allows the purification of soluble and insoluble recombinant proteins to homogeneity with single-step affinity chromatography using immobilized glutathione and metal chelating matrix, respectively. The principle and availability of this new expression system was respectively tested with the purification of a soluble and insoluble recombinant fusion protein containing 24 and 75 amino acids of the human thrombomodulin.     

Simple purification ofEscherichia coli-derived recombinant human interleukin-2 expressed with N-terminus fusion of glucagon

Simple procedures have been devised for purifying recombinant human interleukin-2 (hIL-2), which was expressed inEscherichia coli using sequences of glucagon molecules and enterokinase cleavage site as an N-terminus fusion partner. The insoluble aggregates of recombinant fusion protein produced inE. coli cytoplasm were easily dissolved by simple alkaline pH shift (8→12→8). Following enterokinase cleavage, the recombinant hIL-2 was finally purified by one-step reversed-phase HPLC with high purity. The ease and high efficiency of this simple purification process seem to mainly result from the role of used glucagon fusion partner, which could be applied to the production of other therapeutically important proteins.     

Protein expression strategies for identification of novel target proteins

Identification of new target proteins is a novel paradigm in drug discovery. A major bottleneck of this strategy is the rapid and simultaneous expression of proteins from differential gene expression to identify eligible candidates. By searching for a generic system enabling high throughput expression analysis and purification of unknown cDNAs, we evaluated the YEpFLAG-1 yeast expression system. We have selected cDNAs encoding model proteins (eukaryotic initiation factor-5A [eIF-5A] and Homo sapiens differentiation-dependent protein-A4) and cDNA encoding an unknown protein (UP-1) for overexpression in Saccharomyces cerevisiae using fusions with a peptide that changes its conformation in the presence of Ca2+ ions, the FLAG tag (Eastman Kodak, Rochester, NY). The cDNAs encoding unknown proteins originating from a directionally cloned cDNA library were expressed in all three possible reading frames. The expressed proteins were detected by an antibody directed against the FLAG tag and/or by antibodies against the model proteins. The alpha-leader sequence, encoding a yeast mating pheromone, upstream of the gene fusion site facilitates secretion into the culture supernatant. EIF-5A could be highly overexpressed and was secreted into the culture supernatant. In contrast, the Homo sapiens differentiation-dependent protein-A4 as well as the protein UP-1, whose cDNA did not match to any known gene, could not be detected in the culture supernatant. The expression product of the correct frame remained in the cells, whereas the FLAG-tagged proteins secreted into the supernatant were short, out-of-frame products. The presence of transmembrane domains or patches of hydrophobic amino acids may preclude secretion of these proteins into the culture supernatant. Subsequently, isolation and purification of the various proteins was accomplished by affinity chromatography or affinity extraction using magnetizable beads coated with the anti-FLAG monoclonal antibody. The purity of isolated proteins was in the range of 90%. In the case of unknown cDNAs, the expression product with the highest molecular mass was assumed to represent the correct reading frame. In summary, we consider the YEpFLAG-1 system to be a very efficient tool to overexpress and isolate recombinant proteins in yeast. The expression system enables high throughput production and purification of proteins under physiological conditions, and allows miniaturization into microtiter formats.     

Fusion expression of mutated cecropin CMIV in E. coli

A cDNA coding mutated cecropin CMIV fromBombyx mori was synthesized according to its amino acid sequence usingE. coli biased codons. The gene was cloned into the fusion expression vector pEZZ318 and was expressed inE. coli HB101. The fusion protein produced was purified by affinity chromatography to yield 26 mg/L fusion product. The anti-bacterial activities of recombinant cecropin CMIV were recovered after cleavage by chemical method.     

Evaluation of a Recombinant 27-kDa Outer Membrane Protein of Coxiella burnetii as an Immunodiagnostic Reagent

The 27-kDa outer membrane protein from eight strains of Coxiella burnetii was expressed in the pET-21c protein expression system. Two fusion proteins with molecular masses of 30 and 32 kDa were evident in all eight of the recombinants by SDS-PAGE and immunoblotting. A protein having an approximate size of 30 kDa was purified from the Escherichia coli lysates by one-step affinity purification. The utility of the purified recombinant protein in ELISA was also evaluated by testing its reactivity with human sera and comparing this reactivity with that of Nine Mile phase II antigen. All of the 40 IF-positive serum samples were ELISA-positive for both the Nine Mile phase II and recombinant antigens, and negative serum controls were negative for both antigens. These results suggest that ELISA with the 27-kDa recombinant antigen is a sensitive and specific method for detecting anti-C. burnetii antibodies in human sera.     

Expression of foreign proteins in<Emphasis Type="Italic"> Escherichia coli</Emphasis> by fusing with an archaeal FK506 binding protein

Improper protein-folding often results in inclusion-body formation in a protein expression system using Escherichia coli. To express such proteins in the soluble fraction of E. coli cytoplasm, we developed an expression system by fusing the target protein with an archaeal FK506 binding protein (FKBP). It has been reported that an archaeal FKBP from a hyperthermophilic archaeon, Thermococcus sp. KS-1 (TcFKBP18), possesses not only peptidyl–prolyl cis–trans isomerase activity, but also chaperone-like activity to enhance the refolding yield of an unfolded protein by suppressing irreversible protein aggregation. To study the effect of this fusion strategy with FKBP on the expression of foreign protein in E. coli, a putative rhodanese (thiosulfate sulfurtransferase) from a hyperthermophilic archaeon and two mouse antibody fragments were used as model target proteins. When they were expressed alone in E. coli, they formed insoluble aggregates. Their genes were designed to be expressed as a fusion protein by connecting them to the C-terminal end of TcFKBP18 with an oligopeptide containing a thrombin cleavage site. By fusing TcFKBP18, the expression of the target protein in the soluble fraction was significantly increased. The percentage of the soluble form in the expressed protein reached 10–28% of the host soluble proteins. After purification and protease digestion of the expressed antibody fragment–TcFKBP18 fusion protein, the cleaved antibody fragment (single-chain Fv) showed specific binding to the antigen in ELISA. This indicated that the expressed antibody fragment properly folded to the active form.     

Expression, Purification, and Characterization of a Catalytically Active Human Cytochrome P450 1A2:Rat NADPH-Cytochrome P450 Reductase Fusion Protein

An enzymatically active human cytochrome P450 (P450) 1A2:rat NADPH-P450 reductase fusion protein was purified and partially characterized following heterologous expression inEscherichia coli. A cDNA was engineered to include the coding sequence for human P450 1A2 at its 5′ end (up to but not including the stop codon) fused in-frame to the coding sequence for a truncated (soluble) rat NADPH-P450 reductase at its 3′ end via an oligonucleotide sequence encoding the hydrophilic dipeptide Ser–Thr. This fusion plasmid was expressed inE. coliand the recombinant protein was purified from the detergent-solubilized membrane fraction via sequential DEAE, ADP–agarose, and hydroxylapatite chromatographies. The purified protein has the spectral characteristics of human P450 1A2 and cytochromecreduction activity comparable to rabbit NADPH-P450 reductase. The fusion protein catalyzed 7-ethoxyresorufinO-deethylation and phenacetinO-deethylation to appreciable levels in the presence of NADPH and phospholipid. While these activities were comparable to those of other such P450:NADPH-P450 reductase fusion proteins, they were lower than those of the system reconstituted from its individual hemoprotein and flavoprotein components. Nevertheless, the production of a functional, catalytically self-sufficient monooxygenase inE. colienhances the prospect of using bacterial systems for production and characterization of human P450 drug metabolites as well as for biodegradation of chemicals in the environment.     

Production of engineered IgM-binding single-chain antibodies inEscherichia coli

Summary Two single-chain antibodies were engineered and tested as novel binding proteins with specificity for immunoglobulin M. Genes for the two single-chain Fv proteins were assembled from the variable light chain cDNA and variable heavy chain cDNA of monoclonal antibodies DA4.4 and Bet 2, which specifically bind human IgM and mouse IgM, respectively. Both single-chain Fv proteins were designed with a 14-amino acid linker which bridged the variable light chain and variable heavy chain domains. The two proteins were expressed inEscherichia coli, purified and assayed for IgM-binding activity. Both proteins demonstrate a binding specificity for their corresponding IgM which is similar to the monoclonal antibodies from which they were derived. These small IgM-binding proteins may have applications in the investigation of the immune response and in the detection and purification of monoclonal antibodies, cell-associated antibodies, and IgM from serum.     

Continuous production of restriction endonucleases: continuous two-stage cultivation with E. coli JM103; continuous cell disintegration and purification by affinity chromatography

The optimization of the production of recombinant DNA-derived proteins in Escherichia coli was investigated. We chose restriction endonucleases EcoRI and EcoRV from E. coli as model proteins, despite the observation that overproduction can result in a toxic effect to the cells. The enzymes were expressed as fusion proteins consisting of protein A from Staphylococcus aureus and the desired enzyme in order to facilitate purification. The expression of the fusion protein was induced by a temperature shift using the p_R promoter of phage lambda regulated by the repressor plasmid pRK248cI. Data from batch fermentations provided the basis for planning a continuous two-stage fermentation. The EcoRI enzyme activity was investigated as a function of the induction time after cell disintegration and allowed an estimation of yield of the continuous culture. Plasmid instability, which was only observed under continuous conditions, could be prevented by adding tetracycline (resistance of the repressor plasmid) to the medium. We established a continuous cell disintegration system and purified the fusion protein semicontinuously by affinity chromatography. The biological activity of the fusion protein was the same as the native endonuclease so there was no need for cleavage of the fusion protein and the product could be used without further processing.Correspondence to: K. Schügerl     

Fusion expression of mutated cecropin CMIV inE. coli

A cDNA coding mutated cecropin CMIV fromBombyx mori was synthesized according to its amino acid sequence usingE. coli biased codons. The gene was cloned into the fusion expression vector pEZZ318 and was expressed inE. coli HB101. The fusion protein produced was purified by affinity chromatography to yield 26 mg/L fusion product. The anti-bacterial activities of recombinant cecropin CMIV were recovered after cleavage by chemical method.     

Cloning, Overexpression and Purification of Bacillus subtilis Elongation Factor Tu in Escherichia coli

To establish the overexpression and one-step purification system of Bacillus subtilis elongation factor-Tu (EF-Tu), the EF-Tu gene was amplified with or without own ribosome binding site (rbs) by PCR and the only PCR product without rbs was subcloned successfully. For the expression of the EF-Tu gene cloned after PCR amplification, a constitutive expression system and inducible expression system with His₆ tag at N-terminus or C-terminus, or glutathione-S-transferase (GST) fusion system were examined in E. coli and B. subtilis. Except GST fusion system in E. coli, however, all other trials were unsuccessful at the step of plasmid construction for the EF-Tu expression. The GST/EF-Tu fusion proteins were highly expressed by IPTG induction and obtained as both soluble and insoluble form. From the soluble GST/EF-Tu fusion protein, EF-Tu was obtained to near homogeneity by one-step purification with glutathione-sepharose affinity column chromatography followed by factor Xa treatment. The purified EF-Tu showed high GDP binding activity. These results indicate that the GST/EF-Tu fusion system is favorable to overexpression and purification of B. subtilis EF-Tu.     

CBM21 starch-binding domain: A new purification tag for recombinant protein engineering

The use of protein fusion tag technology simplifies and facilitates purification of recombinant proteins. In this article, we have found that the starch-binding domain derived from Rhizopus oryzae glucoamylase (RoSBD), a member of carbohydrate-binding module family 21 (CBM21) with raw starch-binding activity, is favorable to be applied as an affinity tag for fusion protein engineering and purification in Escherichia coli and Pichia pastoris systems. To determine suitable spatial arrangement of RoSBD as a fusion handle, enhanced green fluorescent protein (eGFP) was fused to either the N- or C-terminus of the SBD, expressed by E. coli, and purified for yield assessment and functional analysis. Binding assays showed that the ligand-binding capacity was fully retained when the RoSBD was engineered at either the N-terminal or the C-terminal end. Similar results have been obtained with the RoSBD-conjugated phytase secreted by P. pastoris. The effective adsorption onto raw starch and low cost of starch make RoSBD practically applicable in terms of development of a new affinity fusion tag for recombinant protein engineering in an economic manner.     

Rational recombinant XMRV antigen preparation and bead coupling for multiplex serology in a suspension array

Diagnosis of infectious diseases often requires demonstration of antibodies to the microbe (serology). A large set of antigens, covering viruses, bacteria, fungi and parasites may be needed. Recombinant proteins have a prime role in serological tests. Suspension arrays offer high throughput for simultaneous measurement of many different antibodies. We here describe a rational process for preparation, purification and coupling to beads of recombinant proteins prepared in Escherichia coli derivate Origami B, to be used in a serological Luminex suspension array. All six Gag and Env proteins (p10, p12, p15, p30, gp70 and p15E), from the xenotropic murine leukemia virus-related virus (XMRV), were prepared, allowing the creation of a multiepitope XMRV antibody assay. The procedure is generic and allows production of protein antigens ready for serological testing in a few working days. Instability and aggregation problems were circumvented by expression of viral proteins fused to a carrier protein (thioredoxin A; TrxA), purification via inclusion body formation, urea solubilization, His tag affinity chromatography and direct covalent coupling to microspheres without removal of the elution buffer. The yield of one preparation (2–10 mg fusion protein per 100 ml culture) was enough for 20–100 coupling reactions, sufficing for tests of many tens of thousands of sera. False serological positivity due to antibodies binding to TrxA and to traces of E. coli proteins remaining in the preparation could be reduced by preabsorption of sera with free TrxA and E. coli extract. The recombinant antigens were evaluated using anti-XMRV antibodies. Although hybrid proteins expressed in E. coli in this way will not have the entire tertiary structure and posttranslational modifications of the native proteins, they contain a large subset of the epitopes associated with them. The described strategy is simple, quick, efficient and cheap. It should be applicable for suspension array serology in general.     

AtJ1, a mitochondrial homologue of theEscherichia coli DnaJ protein

The nucleotide sequence of a cDNA clone fromArabidopsis thaliana ecotype Columbia was determined, and the corresponding amino sequence deduced. The open reading frame encodes a protein, AtJ1, of 368 residues with a molecular mass of 41 471 Da and an isoelectric point of 9.2. The predicted sequence contains regions homologous to the J- and cysteine-rich domains ofEscherichia coli DnaJ, but the glycine/phenylalanine-rich region is not present. Based upon Southern analysis,Arabidopsis appears to have a singleatJ1 structural gene. A single species of mRNA, of 1.5 kb, was detected whenArabidopsis poly(A)⁺ RNA was hybridized with theatJ1 cDNA. The function ofatJ1 was tested by complementation of adnaJ deletion mutant ofE. coli, allowing growth in minimal medium at 44°C. The AtJ1 protein was expressed inE. coli as a fusion with the maltose binding protein. This fusion protein was purified by amylose affinity chromatography, then cleaved by digestion with the activated factor X protease. The recombinant AtJ1 protein was purified to electrophoretic homogeneity.In vitro, recombinant AtJ1 stimulated the ATPase activity of bothE. coli DnaK and maize endosperm cytoplasmic Stress70. The deduced amino acid sequence of AtJ1 contains a potential mitochondrial targeting sequence at the N-terminus. Radioactive recombinant AtJ1 was synthesized inE. coli and purified. When the labeled protein was incubated with intact pea cotyledon mitochondria, it was imported and proteolytically processed in a reaction that depended upon an energized mitochondrial membrane.Abbreviations MBP maltose binding protein - PCR polymerase chain reaction - Stress70c the cytosolic member of the 70 kDA family of stress-related proteins     

Overexpression and Purification of an Immunologically Reactive His–BIV Capsid Fusion Protein

The gene of the capsid protein of bovine immunodeficiency virus (BIV) was linked to a sequence encoding for six histidines and expressed as the (His)₆p26 capsid fusion protein. The fusion protein was strongly expressed as both soluble and insoluble forms after induction by isopropylthio-β- -galactoside. Purification was based on interaction of the hexa-histidine polypeptide with metal ions. Expression could represent 11% of the total protein inEscherichia coli,allowing more than 20 mg of highly purified protein to be obtained per liter of bacterial culture. The (His)₆p26 capsid fusion protein purified by immobilized metal affinity chromatography reacted specifically in Western blot with sera from cattle experimentally infected by BIV, as well as with two monoclonal antibodies directed against different epitopes of the Gag protein. The ease of expression, purification, and specificity of this fusion protein should permit a thorough study of prevalence of BIV infection in large-scale serological studies of field samples.     

Isolation and characterisation of a NBS-LRR resistance gene analogue from pearl millet

Plant resistance (R) proteins belonging to nucleotide-binding site–leucine-rich repeat (NBS–LRR) family are mainly involved in recognition of effectors secreted by pathogens. Pearl millet [Pennisetum glaucum (L.) R.Br] is one of the most drought tolerant cereals, staple food crop of the semi-arid tropics but is highly susceptible to the downy mildew disease caused by oomycetous Sclerospora graminicola (Sacc) schroet. Earlier studies have identified several resistance gene analogues (RGAs) in pearl millet which may be involved in resistance against downy mildew. Of these, a clone RGPM213 was shown to have more than 60% identity with R-proteins coding for NBS–LRR-like protein kinase. The exact nature and function of the R-protein encoded by this gene was not known. In the present study, the cDNA of RGPM213 encompassing NBS–LRR region was inserted into an expression vector pRSET-A and transformed into BL21 E.coli cells. The expressed recombinant fusion protein with a His tag was purified using nickel affinity purification and it had a molecular weight of 35 kDa on SDS-PAGE. Immunoaffinity purification using antibodies raised against this recombinant R-protein identified two proteins of molecular weights 55 kDa and 66 kDa from pearl millet seedling extracts. Peptide mass fingerprinting of these proteins followed by homology search in database revealed similarity of the 55 kDa protein with a protein kinase from Brassica oleracia containing serine/ threonine kinase domain.