A functional raw starch-binding domain of barley alpha-amylase expressed in Escherichia coli

The mature form of barley seed low-pI -amylase (BAA1) possesses a raw starch-binding site in addition to the catalytic site. A truncated cDNA encoding the C-terminal region (aa 281–414) and containing the proposed raw starch-binding domain (SBD) but lacking Trp278/Trp279, a previously proposed starch granule-binding site, was synthesized via PCR and expressed in Escherichia coli as an N-terminal His-Tag fusion protein. SBD was produced in the form of insoluble inclusion bodies that were extracted with urea and successfully refolded into a soluble form via dialysis. To determine binding, SBD was purified by affinity chromatography with cycloheptaamylose as ligand cross-linked to Sepharose. This work demonstrates that a SBD is located in the C-terminal region and retains sufficient function in the absence of the N-terminal, catalytic, and Trp278/279 regions.     

Molecular cloning and expression of the pyranose 2-oxidase cDNA from<Emphasis Type="Italic"> Trametes ochracea</Emphasis> MB49 in<Emphasis Type="Italic"> Escherichia coli</Emphasis>

A cDNA of a structural gene encoding pyranose 2-oxidase (P2O) from Trametes ochracea strain MB49 was cloned into Escherichia coli strain BL21(DE3) on a multicopy plasmid under the control of the trc promoter. Synthesis of P2O was studied in batch cultures in LB or M9-based mineral medium at 28°C. While there was a low specific activity of P2O in LB medium, the enzyme was synthesised constitutively in mineral medium and represented 3% of the cell soluble protein (0.3 U mg^–1). The effect of isopropyl -d-thiogalactoside on the expression of P2O was studied in mineral medium at 25 and 28°C. The synthesis of P2O at 28°C corresponded to 39% of the cell soluble protein but the major portion of P2O (93%) was in the form of non-active inclusion bodies (activity of P2O equalled 0.19 U mg^–1). At 25°C, the amount of P2O represented 14% of the cell soluble protein and the activity of P2O was 1.1 U mg^–1. The soluble enzyme represented 70% of the total amount of P2O.     

Bacterial expression and purification of human papillomavirus type 18 L1

The human papillomavirus (HPV) 18 L1 gene, which encodes the L1 major capsid protein, was isolated from a female patient in Pusan, Korea Republic and was cloned into pGEX-4T-1 vector. The HPV-18L1 gene was expressed in Escherichia coli as a fusion protein with a glutathione-S-transferase (GST) tag. The soluble recombinant fusion protein, GST-18 L1 fusion, was isolated to high purity. HPV-18 L1 was purified from the GST-18 L1 fusant after biotinylated thrombin cleavage, and then the treated thrombin was removed serially using streptavidin conjugated resin. The purified HPV-18 L1 was confirmed by western blotting using a rabbit anti-denatured papillomavirus polyclonal antibody. The virus-like particles (VLP) from the purified full-length 18 L1 protein without any extra amino acid sequences was observed through the analysis of the electron microscope. This is the first study to report the expression and purification of HPV-18 L1 in E. coli. This expression and purification system offers a simple method of expressing and purifying HPV L1 protein, and could potentially be an effective route for the development and manufacturing of highly purified HPV-18 L1-based cervical cancer vaccines.     

Inefficient processing of human protein C in the mouse mammary gland

Vitamin K-dependent plasma protein, human Protein C (HPC) has been expressed in transgenic mice, using a 4.2kb mouse whey acidic protein (WAP) promoter, 9.0 kb HPC gene and 0.4 kb 3flanking sequences. Expression was mammary gland-specific and the recombinant human Protein C (rHPC) was detected in milk at concentrations of 0.1 to 0.7mg ml^–1. SDS-PAGE revealed that the single, heavy and light chains of rHPC migrated with increased electrophoretic mobility, as compared to HPC. Enzymatic deglycosylation showed that these molecular weight disparities are in part due to differential glycosylation. The substantial increase observed in the amount of single chain protein, as well as the presence of the propeptide attached to 20–30% of rHPC, suggest that mouse mammary epithelial cells are not capable of efficient proteolytic processing of rHPC. TheK _m of purified rHPC for the S-2366 synthetic substrate was similar to that of plasma-derived HPC, while the specific activity was about 42–77%. Amino acid sequence analyses and low anticoagulant activity of purified rHPC suggest that -carboxylation of rHPC is insufficient. These results show that proteolytic processing and -carboxylation can be limiting events in the overexpression of fully biologically active rHPC in the mouse mammary gland.     

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.     

Cloning and expression of soluble recombinant protein comprising the extracellular domain of the human type I interferon receptor 2c subunit (IFNAR-2c) in E. coli

An optimized procedure was developed for production of the extracellular domain encoding amino acids 1–243 of the human type I interferon receptor 2c subunit (IFNAR-2c) as a fusion protein with glutathione S-transferase (GST-IFNAR2cEC) in E. coli to obtain active, soluble protein. Induction of protein expression at 37 °C resulted in a formation of inclusion body. Co-expression with bacterial chaperones, GroEL and GroES, failed to support the folding of GST-IFNAR2cEc under IPTG induction at 37 °C. Expression induced at 25 °C decreased the inclusion body formation up to 62% and cell disruption by a French press provided higher recovery of the recombinant protein than cell disruption by sonication.     

Immuno-rolling circle amplification using a multibinding fusion protein

Ultrasensitive detection of specific, low level proteins in body fluids is particularly challenging. Owing to the extreme sensitivity of the polymerase chain reaction step, the requirements for immuno-rolling circle amplification (immuno-RCA) are much more stringent than for conventional ELISA. Here, we report the development of a rolling circle amplification procedure using multibinding fusion protein to enhance signals of immuno-RCA to detect a cancer biomarker, α-fetoprotein (AFP). We successfully avoid the covalent linkage between antibody and DNA or antibody and biotin/streptavidin by introducing a new genetically engineered fusion protein which contains the C2 domain of protein G and biotin acceptor peptide (BAP) which is intended to maintain the biological activity of the antibody. The purified fusion protein retained its binding affinity with IgG and streptavidin after efficient expression in Escherichia coli. Immuno-RCA in combination with BAP-C2 specifically and sensitively detected AFP in a microplate format. Therefore, the sensitivity and convenient nature of this method should contribute to effective signal enhancement in immunoassays for cancer biomarker detection.     

A two-site immunometric assay for the detection of the expression level of α2(C2)-adrenergic receptor produced in Escherichia coli

Human ₂(C2)-adrenergic receptor was expressed in Escherichia coli as a fusion protein with Bacillus circulans var. alcalophilus cyclomaltodextrin glucanotransferase. For the determination of the expression level (0.6 mg of solubilized fusion protein l^–1 of E. coli culture), a two-site immunometric assay based on two monoclonal antibodies with different epitopes was developed.     

High-level expression of recombinant human FK-binding protein from a fusion precursor

The human peptidyl-prolyl isomerase FK-binding protein (FKBP) was cloned as a fusion partner with CMP-KDO synthetase (CKS), and the resultant construct was characterized as an improved high-expression source for FKBP. The CKS-FKBP fusion was expressed as a soluble protein at levels approaching 1 gm/L inEscherichia coli fermentations. The fusion protein was purified to near homogeneity by a one-step ammonium sulfate fractionation of whole cell lysate. After selective cleavage, the fusion precursor produced yields approaching 300 mg of purified FKBP per liter of harvested culture, a 30 to 60-fold increase over that observed for a nonfusion construct. Selective cleavage of the fusion partners was accomplished using either hydroxylamine or specific, limited proteolysis. Once separated from the CKS fusion partner, the FKBP was isolated in a single step by either reversed-phase HPLC or chromatography on Q-Sepharose. For comparison of physical and chemical properties, a nonfusion construct of recombinant human FKBP was expressed inE. coli and isolated. The purified FKBPs exhibited expected SDS-PAGE molecular weights and N-terminal sequences. The proteins had similar proton NMR spectra and binding to [³H]FK-506. The fusion construct, CKS-FKBP, was also found to bind [³H]FK-506. These data indicate that FKBP fused to the C-terminus of CKS folds independently of the fusion partner and suggests the fused FKBP adopts a conformation resembling that of the native protein.     

Efficient expression and characterization of hepatitis B virus preS2 antigen in Escherichia coli

The complete (encoding 55 amino acids, aa) or partial (encoding aa 1–26) preS2 region gene of hepatitis B virus (HBV) was fused to the 3-end of glutathion-S-transferase (GST) gene and expressed under the control of the inducible tac promoter in Escherichia coli at 37 °C. The fusion protein with the complete preS2 region was moderately expressed (8%) while the protein with the N-terminal 26 aa was expressed at a higher level, yielding about 20% of the total cellular proteins. The GST-preS2 (aa 1–26) protein, which contains the immunodominant epitope, was produced form the soluble protein fraction of the recombinant bacteria and purified by affinity chromatography using glutathione-agarose column. The purified preS2 fusion protein showed the antigenicity of preS2, as assessed by indirect and competitive ELISAs.     

Recombinant proprotein convertase 4 (PC4) from Leishmania tarentolae expression system: purification, biochemical study and inhibitor design

Proprotein convertase 4 (PC4) is a member of Ca²⁺-dependent mammalian subtilases called Proprotein convertases (PCs) or Proprotein convertases subtilisin kexin (PCSK). PC4 plays a key role in mammalian fertilization, sperm maturation and sperm–egg fusion. Full length and C-terminal truncated rPC4 have been expressed using Leishmania tarentolae expression system. Secreted soluble enzyme was recovered in good yield from concentrate medium and purified by DEAE anion exchange and arginine–agarose column chromatographies. This is the first attempt to produce rec (recombinant) PC4 by Leishmania expression system in reasonably pure and enzymatically active form. The eluted fraction contained PC4 protein as confirmed by immunoreactivity using PC4-specific antibodies. Two protein bands at 62, 53 kDa in SDS–PAGE were attributed to C-terminal truncated PC4 forms. The fraction displayed strong protease activity towards fluorogenic Boc-RVRR-MCA and various intramolecularly quenched peptides derived from PC4-substrates. It also cleaved proIGF-2 to produce active IGF-2 confirming its role in this maturation process. Moreover PC4-mediated proteolysis was efficiently blocked by a newly designed prodomain rPC4^101–116 peptide with IC₅₀ in low μM level. Similar but more potent PC4-inhibitory activity with K_i in low nM range was observed with the tetrapeptide chloromethyl ketones, Dec-RVKR/K-cmk (chloromethyl ketone). The study showed that such PC4 inhibitors may find potential therapeutic and clinical applications in male fertility.     

Fusion protein of the hyaluronan binding domain from human TSG-6 with luciferase for assay of hyaluronan

The gene expression plasmid, pET-Lmluc, for the fusion protein of the hyaluronan binding domain from human TSG-6 [product of tumor necrosis factor (TNF)-stimulated gene-6] and luciferase from Renilla reniformis was constructed. The fused gene was expressed in Escherichia coli and the resulted insoluble Lm-luc fusion protein was purified and refolded to recover both the hyaluronan binding capability and the luciferase activity. Hyaluronan as low as 1 ng ml^–1 was detected by using the indirect enzymatic immunological assay with the refolded Lm-luc fusion protein.     

Molecular characterization of Alg8, a putative glycosyltransferase, involved in alginate polymerisation

The topology of Alg8, the proposed catalytic subunit of the alginate polymerase, was assessed using PhoA and LacZ fusion protein analysis. This analysis suggested that the periplasmic loop comprises only three amino acid residues with the adjacent transmembrane helices at positions 361–387 and 393–416. Accordingly, the extended cytosolic loop could be located at positions 71–361 and was proposed to contain important catalytic residues. Further experimental evidence for this cytosolic domain was obtained by independently demonstrating this protein region as purified soluble protein domain. The soluble protein domain was identified by MALDI-TOF/MS and presumably represents the cytosolic catalytic domain of Alg8. Site-directed mutagenesis of 11 conserved residues in the cytosolic loop showed that D-188/D-190 (DXD motif), D-295/D-296 (acid–base catalysts) and K-297 were each essential for in vivo polymerase activity, whereas D-179/D-181 (DXD motif), C-244, R-263, D-279, and E-282 were not directly involved in the polymerisation reaction. The role of these amino acid residues with respect to the catalysed alginate polymerisation reaction was discussed with the aid of the recently developed structural model of Alg8.     

Factors that influence the extracellular expression of streptavidin in <Emphasis Type="Italic">Escherichia coli</Emphasis> using a bacteriocin release protein

Aiming to increase production of recombinant streptavidin in Escherichia coli, the effect of different leader sequences, different promoter strengths of the bacteriocin release protein (kil), host strain and medium composition on the expression and secretion into the medium was investigated. Expression vectors containing an expression or secretion unit were constructed with different combinations of leader sequence for the streptavidin gene and promoters for the kil gene and streptavidin gene. Results showed that a high-level extracellular production of streptavidin could be accomplished with E. coli BL21(DE3) by using the leader sequence of the phoA gene, a strong stationary-phase promoter for the kil gene and supplementation of the medium by glycine. Using a stationary-phase promoter for the expression of streptavidin had a negative effect.     

Construction of bifunctional fusion proteins consisting of duck BAFF and EGFP

We constructed fusion proteins consisting of fluorescence-enhanced green fluorescent protein (EGFP) and soluble domain of duck B-cell-activating factor of the TNF family (dsBAFF). The soluble EGFP/dsBAFF was efficiently expressed in Escherichia coli BL 21 (DE3) and was purified in milligram amounts using metal chellate affinity chromatography. The fusion protein exhibited similar fluorescence spectra with free EGFP and promoted the survival of duck bursal B cells in vitro as well as dsBAFF. EGFP/dsBAFF has shown specific binding to duck BAFF receptors positive-cells and the stained cells could be analyzed with flow cytometry. Thus, the fusion protein represents a readily obtainable source of biologically active dsBAFF that may prove useful in further studies on duck BAFF and its receptors.     

Construction and application of new Corynebacterium glutamicum vectors

The construction of new Corynebacterium glutamicum/Escherichia coli shuttle vectors with improved cloning properties and an increased chloramphenicol resistance (50 g ml^–1 MIC) is described. A modified glnB gene encoding a Strep-tag II domain modified P_II protein was expressed in C. glutamicum and streptavidin affinity chromatography was used to purify this protein.     

Expression and purification of human respiratory syncytial virus recombinant fusion protein

The Human Respiratory Syncytial Virus (HRSV) fusion protein (F) was expressed in Escherichia coli BL21A using the pET28a vector at 37 °C. The protein was purified from the soluble fraction using affinity resin. The structural quality of the recombinant fusion protein and the estimation of its secondary structure were obtained by circular dichroism. Structural models of the fusion protein presented 46% of the helices in agreement with the spectra by circular dichroism analysis. There are only few studies that succeeded in expressing the HRSV fusion protein in bacteria. This is a report on human fusion protein expression in E. coli and structure analysis, representing a step forward in the development of fusion protein F inhibitors and the production of antibodies.     

A novel purification of ferric citrate receptor (FecA) from Escherichia coli UT5600 and further characterization of its binding activity

In our earlier paper, it was demonstrated that the FecA receptor protein from Escherichia coli UT5600/pBB2 (leu ^–, proC ^–, trpE ^–, entA ^–, rpsl ^–, (ompT-fepA)^–/Ampr, fepA) binds with ferric enterobactin. In order to explore this further the outer membrane receptor protein, FecA, has been isolated from UT5600 (fepA ^–) and purified to homogeneity by DE-52-cellulose anion exchange chromatography followed by MonoPFPLC chromatofocusing. Partially purified FecA and homogeneous FecA show binding activity to [55Fe]ferric enterobactin and the binding is specific. Binding activity of FecA can be enhanced by ferric citrate. Lipopolysaccharide-free FecA as ascertained by silver staining and the endotoxin test still retains the same activity. In vivo uptake studies using different strains of E. coli suggest that FecA in E. coli plays an important role in ferrienterobactin transport.     

Investigation of factors influencing production of the monocyclic carotenoid torulene in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Factors influencing production of the monocyclic carotenoid torulene in recombinant Escherichia coli were investigated by modulating enzyme expression level, culture conditions, and engineering of the isoprenoid precursor pathway. The gene dosage of in vitro evolved lycopene cyclase crtY2 significantly changed the carotenoid profile. A culture temperature of 28°C showed better production of torulene than 37°C while initial culture pH had no significant effect on torulene production. Glucose-containing LB, 2×YT, TB and MR media significantly repressed the production of torulene, and the other carotenoids lycopene, tetradehydrolycopene, and -carotene, in E. coli. In contrast, glycerol-containing LB, 2×YT, TB, and MR media enhanced torulene production. Overexpression of dxs, dxr, idi and/or ispA, individually and combinatorially, enhanced torulene production up to 3.1–3.3 fold. High torulene production was observed in a high dissolved oxygen level bioreactor in TB and MR media containing glycerol. Lycopene was efficiently converted into torulene during aerobic cultures, indicating that the engineered torulene synthesis pathway is well coordinated, and maintains the functionality and integrity of the carotenogenic enzyme complex.     

Tissue-specific regulation of BiP genes: a cis-acting regulatory domain is required for BiP promoter activity in plant meristems

The binding protein BiP is an endoplasmic reticulum (ER)-resident member of the HSP70 stress-related protein family, which is essential for the constitutive function of the ER. In addition to responding to a variety of environmental stimuli, plant BiP exhibits a tissue-specific regulation. We have isolated two soybean BiP genomic clones, designated gsBiP6 and gsBiP9, and different extensions of their 5 flanking sequences were fused to -glucuronidase (GUS) reporter gene and introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Transgenic plants displayed prominent GUS activity in the vascular bundles of roots and shoots as well as in regions of intense cell division, such as procambial region and apical meristems. Promoter deletion analyses identified two cis-regulatory functional domains that are important for the spatially-regulated activation of BiP expression under normal plant development. While an AT-rich enhancer-like sequence, designated cis-acting regulatory domain 1, CRD1 (–358 to –211, on gsBiP6), activated expression of the BiP minimal promoter in all organs analyzed, BiP promoter activity in meristematic tissues and phloem cells required the presence of a second activating domain, CRD2 (–211 to –80). Apparently, the CRD2 sequence also harbors negative cis-acting elements, because removal of this region caused activation of gsBiP6 promoter in parenchymatic xylem rays. These results suggest that the tissue-specific control of BiP gene expression requires a complex integration of multiple cis-acting regulatory elements on the promoter.