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.     

The ;heavy' subunit of the photosynthetic reaction centre from Rhodopseudomonas viridis: isolation of the gene, nucleotide and amino acid sequence

The gene coding for the `heavy' subunit of the photosynthetic reaction centre from Rhodopseudomonas viridis was isolated in an expression vector. Expression of the heavy subunit in Escherichia coli was detected with antibodies raised against crystalline reaction centres. The entire subunit, and not a fusion protein, was expressed in E. coli. The protein coding region of the gene was sequenced and the amino acid sequence derived. Part of the amino acid sequence was confirmed by chemical sequence analysis of the protein. The heavy subunit consists of 258 amino acids and its mol. wt. is 28 345. It possesses one membrane-spanning α-helical segment, as was revealed by the concomitant X-ray structure analysis.     

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.     

The DsbA signal peptide-mediated secretion of a highly efficient raw-starch-digesting,recombinant α-amylase from Bacillus licheniformis ATCC 9945a

In this study, a new approach for extracellular production of recombinant α-amylase in Escherichia coli was investigated. A gene encoding a highly efficient raw-starch-digesting α-amylase from Bacillus licheniformis ATCC 9945a was cloned and expressed in E. coli. The gene encoding mature α-amylase was cloned into the pDAss expression vector, and secretion of the gene product was regulated by fusion to the signal peptide of DsbA, a well-characterized E. coli periplasmic protein. E. coli BL21 (DE3) carrying pDAss vector containing amylase gene had approximately 2.5-fold higher volumetric enzyme productivity than the natural system. The recombinant enzyme showed higher efficiency for digesting diverse raw starches when compared with the native enzyme and was similar to commercial α-amylase in its ability to hydrolyze raw starches. The properties of the recombinant enzyme demonstrate the potential of the DsbA signal peptide approach for the secretory production of the fully active, industrially important recombinant enzyme.     

Somatic homologous recombination in planta: The recombination frequency is dependent on the allelic state of recombining sequences and may be influenced by genomic position effects

The Escherichia coli sodA gene encoding the antioxidant enzyme Mn-containing superoxide dismutase (MnSOD), was cloned in the expression vector pMG36e. This vector has a multiple cloning site down-stream of a promoter and Shine-Dalgarno sequences derived from Lactococcus. The protein-coding region of sodA from E. coli was amplified by the polymerase chain reaction, using a thermocycler and Taq DNA polymerase before cloning into pMG36e. When introduced into E. coli, the recombinant plasmid expressed the predicted fusion protein, both in the presence and absence of oxygen. The expression of the fusion protein in E. coli was verified by SOD assays, activity gels and Western blots. The recombinant plasmid was also introduced into Lactococcus lactis, which contains a resident SOD, and into Lactobacillus gasseri, which is devoid of SOD. Transformed lactococci expressed an active SodA fusion protein plus an active hybrid protein composed of subunits of the Lactococcus and the recombinant E. coli enzymes. Transformants of L. gasseri expressed only the fusion SodA protein, which was enzymatically active.     

Bioaccumulation of heavy metals in Escherichia coli expressing an inducible synthetic human metallothionein gene

A synthetic DNA coding for human hepatic metallothionein (HMT), fraction MT-2, has been cloned in the plasmid vector pING 1 and expressed in E. coli. Upon induction with arabinose, an araB′-HMT fusion protein is synthesized. The fusion protein is produced to a level of approximately 8% of the total protein in E. coli, with an apparent half-life of 50 min. Without arabinose, no fusion protein is expressed, demonstrating that the expression system is tightly regulated. In the presence of ¹⁰⁹Cd, the fusion protein binds the metal ion in vitro. Concurrently, a direct correlation is found between the expression in E. coli of the araB′-HMT fusion protein which binds Cd²⁺ in vitro and the bioaccumulation of Cd²⁺ in vivo. The bioaccumulation of Cu⁺ in E. coli, when the fusion protein is produced, has also been established.     

High-level expression of human renin in Escherichia coli

The cDNA sequence for human renin was modified for use in the expression of the mature protein in E. coli. This was accomplished by the removal of the 5′ untranslated region and sequences coding for the signal peptide and a portion of the mature protein. An oligonucleotide linker was inserted which supplied the deleted coding information for the mature protein in a form optimized for translation in E. coli, in addition to an initiation codon. The modified gene was cloned into an expression vector consisting of the promoter from the tryptophan operon of E. coli and trp L Shine-Dalgarno sequence. In an appropriate host strain the expressed protein is the most prominent species present, and accounts for at least 10% of the total cellular protein. The expressed protein was verified to be renin by its molecular weight, ability to bind a renin antibody, and N-terminal amino acid sequence.     

The Klebsiella aerogenes glutamate dehydrogenase (gdhA) gene: cloning, high-level expression and hybrid enzyme formation in Escherichia coli

Summary The NADP-dependent glutamate dehydrogenase gene of Klebsiella aerogenes was cloned in E. coli in the expression plasmid pRK9. The cloned gene shows a high level of expression in E. coli in the hybrid plasmid pKG3 and such expression is independent of the vector promoter, as shown by experiments in which the promoter was deleted. Active hybrid GDH hexamers were shown in cell-free extracts of an E. coli strain carrying cloned gdhA genes of both E. coli and K. aerogenes. The nucleotide sequence of the N-terminal coding region of the K. aerogenes gdhA gene was determined and found to be strongly homologous with that of E. coli.Abbreviations GDH glutamate dehydrogenase - PMS phenazine methosulphate - MTT 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium-bromide - PMSF phenylmethylsulphonylfluoride - SSC standard saline citrate - DTT dithiothreitol - bp base pairs - kbp kilo base pairs - dNTP deoxynucleoside triphosphate     

A Novel Dual Vector Coexpressing PhiX174 Lysis E Gene and Staphylococcal Nuclease A Gene on the Basis of Lambda Promoter pR and pL, Respectively

Bacterial ghost is a novel vaccine platform, and its safe and efficient production depends largely upon a suitable and functional vector. In this study, a series of temperature-inducible plasmids, carrying Phix174 lysis gene E and/or staphylococcal nuclease A (SNA) gene, were constructed and evaluated in Escherichia coli. The results showed that the direct product of SNA (pBV220-SNA) could degrade the plasmid and genomic DNA of E. coli while the fusion product of gene E and partial Cro gene (pKF396M-2) lost the ability to lyse the host strain. The insertion of enhancer T7g10 elements and Shine–Dalgarno box (ESD) between them (pKF396M-3) could resume the function of gene E. Using plasmid pKF396M-4 with gene E and SNA, respectively, under the immediate control of promoter pR and pL, the remnant plasmids and genomic DNA of E. coli were eliminated, and the rates of inactivation increased by two orders of magnitude over that obtained with the exclusive use of E-mediated lysis plasmid. By substituting these two genes with customized multiple cloning sites sequences, the plasmid could be modified to a dual expression vector (pKF396M-5).     

Production and purification of a cecropin family antibacterial peptide,hinnavin II,in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The antibacterial peptide hinnavin II, isolated from the cabbage butterfly Artogeia rapae, is synthesized with an amidated lysine 37 residue at C-terminus. Glycine-extended native hinnavin II (hinnavin II-38-Gly, hin II) gene with 114 bp coding region was cloned in the expression vector pET-32a (+) to construct a fusion expression plasmid and transformed into Escherichia coli BL21 (DE3) pLysS. The recombinant fusion protein Trx-hin II was expressed in soluble form, purified successfully by Ni²⁺-chelating chromatography, and cleaved by enterokinase to release recombinant hin II (rhin II). Purification of the rhin II was achieved by reversed-phase FPLC, and 2.45 mg pure active rhin II was obtained from 800 mL E. coli culture. The molecular mass of the rhin II determined by MALDI-TOF mass spectrometry is consistent with the theoretical molecular mass of 4,195.0 Da. The purified rhin II showed antimicrobial activities against tested E. coli K 12, E. coli BL21 (DE3), Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus. The application of this expression/purification approach represents a fast and efficient method to prepare milligram quantities of hinnavin II in its biologically active form.     

Flow cytometry of Escherichia coli for process monitoring

A laser flow cytometer was used to study different Escherichia coli populations under various cultivation conditions. A host strain E. coli 5K was analyzed for cell size, protein and DNA-content during continuous cultivation. Also, a recombinant E. coli 5K(pHM12) strain (used for the intracellular production of penicillin-G acylase) was studied in regard to gene expression using different cytometric techniques. An argon ion laser (30 mW) and a 100 W high-pressure mercury lamp were used as light source in the cytometer. A new fluorogenic staining technique for intracellular penicillin-G acylase is described.Recombinant E. coli temperature sensitive cells were analyzed for intracellular fusion protein production due to temperature induction.     

Refolding of recombinant Pasteurella haemolytica A1 glycoprotease expressed in an Escherichia coli thioredoxin gene fusion system

Pasteurella haemolytica A1 secretes an O-sialoglycoprotein endopeptidase (EC. 3.4.24.57) (glycoprotease: Gcp) which is specific for O-linked sialoglycoproteins. When the cloned gene is expressed in Escherichia coli, the recombinant glycoprotease (rGcp) is secreted to the peripalsm where it is present as a disulfide-linked aggregate which lacks enzymatic activity. In vitro refolding and activation of rGcp by mammalian protein disulfide isomerase (PDI) or by the E. Coli chaperones (DnaK, DnaJ and GrpE) indicate that the redox environment of rGcp is critical in restoring biological activity. A fusion protein, rTrx-Gcp, was constructed to investigate the role of thioredoxin (E. coli TrxA) in the production of enzymatically active rGcp. This 47 kDa protein was expressed at a high level, in a soluble, monomeric form, in the cytoplasm of E. coli. Cleavage of the fusion protein by enterokinase released the rGcp fragment (35 kDa) with glycoprotease activity. A higher recombinant glycoprotease activity was recoveref after anion exchange chromatography of lystates of E. coli expressing rTrx-Gcp. Thus when E. coli TrxA is combined in a recombinant fusion protein with P. haemolytica A1 Gcp, productive folding of the glycoprotease can occur as a result of the chaperone action of the protein disulfide reductase coupled with its ability to retain the fusion gene product in the E. coli cytopalsm.     

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.     

The Histidyl-tRNA Synthetase from Streptococcus equisimilis: Overexpression in Escherichia coli,Purification, and Characterization

ABSTRACT

We describe the high-level expression of the Streptococcus equisimilis histidyl-tRNA synthetase gene hisS) in Escherichia coli and the purification and characterization of the gene product. Due to a lack of an efficient E. coli ribosome binding sequence in the hisS gene, the coding region was fused in-frame to the expression vector pT7-7, thereby creating a fusion gene construct (pT7-7recIII), which is under the control of a strong bacteriophage T7 promoter. Another construct (pT-7recII) was used for low level expression of the native histidyl-tRNA synthetase (HisRS). The plasmids were electroporated into E. coli HB101, which already contained pGP1-2. After temperature induction, the fusion HisRS, which has an extra 15 amino acids between the initiator Met and the second amino acid, Lys, was expressed at a level of —18% of total cell protein (～50 mg'liter of bacterial culture). The fusion HisRS was purified to >99% by a combination of anion exchange and cation exchange chromatography of the S100 fraction. The predicted MWs of the native and fusion proteins are 47,932 and 49,717, respectively. The mass of the active fusion HisRS was estimated to be 94,000 Da by Sephacryl S-200 gel filtration chromatography and 108,200 Da by nondenaturing PAGE. Both methods show that the funtional enzyme is a dimer of two identical subunits. SDS-PAGE analysis of purified fusion HisRS with or without reduction showed a single band of M_r = 53.7 kDa.     

Production of the human immunoglobulin γ1 chain constant region polypeptides in Escherichia coli

We have determined the nucleotide sequence of the constant region exons of the rearranged human immunoglobulin γ₁ chain gene cloned from a human plasma cell leukemia line, ARH-77. The amino acid sequence deduced from the nucleotide sequence revealed that the allotype of the ARH-77 γ₁ chain was Glm (−1, −2, 3).Recombinant plasmids were then constructed in order to express the human γ₁ chain constant region genes (the Fc region gene and the C_H2-C_H3 domains gene) in Escherichia coli. The human γ₁ chain constant region genes without introns were derived from the genomic gene using synthetic DNA fragments. E. coli carrying each expression plasmid produced antigenically active constant region polypeptides as soluble proteins. In the case of the E. coli-derived Fc region polypeptides, they were generated as monomeric forms in the cytoplasm.     

A bifunctional exoglucanase-endoglucanase fusion protein

A fusion was constructed between the cex gene of Cellulomonas fimi, which encodes an exoglucanase, and the cenA gene of the same organism, which encodes an endoglucanase. The cex-cenA fusion was expressed in Escherichia coli to give a fusion protein with both exoglucanase and endoglucanase activities. The fusion protein, unlike the cex and the cenA gene products from E. coli, did not bind to microcrystalline cellulose, presumably because it lacked an intact substrate-binding region. The fusion protein was exported to the periplasm in E. coli.     

Overproduction of Biologically-active Human Nerve Growth Factor in Escherichia coli

A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E. coli. The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the β-lactamase signal peptide. The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene. E. coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the β-lactamase signal peptide. The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth. This biological activity was comparable to that of authentic mouse NGF. E. coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein. Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells.