Expression in Escherichia coli of chemically synthesized gene for a novel opiate peptide alpha-neo-endorphin

Chemically synthesized alpha-neo-endorphin gene was fused to the Escherichia coli beta-galactosidase gene on the plasmid pKO13. The resulting recombinant DNA was used to transform E. coli cells. Radioimmunoassay for alpha-neo-endorphin in CNBr-treated bacterial cells showed that alpha-neo-endorphin was synthesized at approximately 5 x 10(5) molecules per single E. coli cell. One of the transformants, WA802/p alpha NE2, was used for alpha-neo-endorphin purification. From 10.9 g of wet cells, we isolated 4 mg of chemically pure and biologically active alpha-neo-endorphin.     

Expression in Escherichia coli of a chemically synthesized gene for a "mini-C" analog of human proinsulin

A gene has been constructed which codes for an analog of human proinsulin in which the normal 35-amino acid connecting peptide is replaced by a "mini-C" peptide of six amino acids (Arg-Arg-Gly-Ser-Lys-Arg). The gene, composed of oligonucleotide fragments synthesized by the triester method, was cloned and expressed as a beta-galactosidase hybrid protein. The proinsulin analog was separated from beta-galactosidase by cyanogen bromide cleavage and purified. Controlled disulfide exchange in the S-sulfonate of the analog generated a molecule having high-pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) behavior consistent with a proinsulin-like structure.     

Expression of the chemically synthesized gene for ribonuclease T1 in Escherichia coli using a secretion cloning vector

The gene for ribonuclease T1 from Aspergillus oryzae has been chemically synthesized using the segmental support technique. An Escherichia coli clone producing the ribonuclease at high levels was constructed by linking the gene downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E. coli), using the secretion cloning vector pIN-III-ompA2. This strategy was employed in order to circumvent a possible toxic effect of the gene product on the host cell. Active ribonuclease containing four additional amino acids at the N-terminus could be isolated from the periplasmic fraction of the host. The final yield after purification was 20 mg enzyme/l liquid culture. With respect to immunological, catalytic and specific behaviour, no qualitative differences could be detected between the enzyme from the over-producing E. coli strain and ribonuclease T1 isolated from A. oryzae.     

Expression of chemically synthesized alpha-neo-endorphin gene fused to E. coli alkaline phosphatase.

An alpha-neo-endorphin (alpha NE) gene, which we previously synthesized chemically and inserted into E. coli beta-galactosidase gene of pK013 plasmid, has been excised and fused to E. coli alkaline phosphatase (APase) gene. One of the transformants was named E15/pA alpha NE1. Under the APase gene regulation, APase-alpha NE chimeric protein was expressed at 1.3 X 10(6) molecules per cell, and accounted for about 60% of total cellular proteins. The HPLC pattern of CNBr treated E15/pA alpha NE1 was very simple reflecting the high content of the chimeric protein and low numbers of methionine residues in it. A series of genes encoding APase-alpha NE chimeric proteins in which 30 to 94 C-terminal amino acid residues were replaced by (met)-alpha NE, was cloned in E. coli. Transportation of the chimeric proteins to periplasmic space was studied. All chimeric proteins were apparently processed by signal peptidase but few, if any, was transported to the periplasmic space.     

Chemotactic response of Escherichia coli to chemically synthesized amino acids.

In Escherichia coli, seven of the commonly occurring amino acids are strong attractants: L-aspartate, L-serine, L-glutamate, L-alanine, L-asparagine, glycine, and L-cysteine, in order of decreasing effectiveness. The chemotactic response to each amino acid attractant is mediated by either methyl-accepting chemotaxis protein I or II, but not by both. Seven of the commonly occurring amino acids are repellents. This work was carried out with chemically synthesized amino acids.     

High-level expression in Escherichia coli of a chemically synthesized gene for [Leu-28]echistatin

A gene (Ecs) encoding a platelet aggregation inhibitor, echistatin (Ecs), has been chemically synthesized. Met at position 28 of the native protein was replaced by Leu in the recombinant Ecs. To express this synthetic gene in Escherichia coli, an expression vector, pJC264, was constructed by inserting portions of the E. coli cheB and cheY gene complex into the plasmid pUC13. High-level expression of the synthetic [Leu-28]Ecs was achieved by its fusion with the E. coli cheY gene in the expression vector. Recombinant [Leu-28]Ecs was liberated from the fusion protein by CNBr cleavage at the Met inserted between the CheY protein and [Leu-28]Ecs. The recombinant [Leu-28]Ecs was purified to homogeneity by reverse-phase high-performance liquid chromatography. The refolded [Leu-28]Ecs was identical to native Ecs in inhibiting platelet aggregation, suggesting that Met at position 28 is not essential for the biological activity of this platelet aggregation inhibitor.     

Production of biologically active thymulin in Escherichia coli through expression of a chemically synthesized gene

Summary A synthetic gene coding for thymulin was ligated into an expression vector (pJB 1301) and placed under lac operon control. In the recombinant clones, thymulin was expressed as part of a galactosidase chimeric protein which was then cleaved by cyanogen bromide. Thymulin was purified using various chromatography systems including gel filtration and HPLC, and was detected by radioimmunoassay (RIA). In the biological assay the purified recombinant peptide demonstrated the same zinc dependency as natural thymulin and had the same amino acid composition and primary structure.     

Expression of the chemically synthesized coding region for the cytotoxin alpha-sarcin in Escherichia coli using a secretion cloning vector

The coding region for the cytotoxin alpha-sarcin from Aspergillus giganteus has been chemically synthesized by the ligation of 19 overlapping oligodeoxyribonucleotides. An Escherichia coli clone producing the cytotoxin was constructed by inserting the synthesized gene directly downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E. coli), using the secretion cloning vector pIN-III-OmpA2. The enzyme encoded by the chemically synthesized gene expressed in E. coli displayed properties identical to those of native alpha-sarcin isolated from A. giganteus with respect to its chemistry, antigenicity and ribonucleolytic activity in qualitative assays.     

Expression in Escherichia coli cells of mutant human interferon alpha2

cDNA library was obtained from mRNA isolated from human leukocytes induced by Newcastle disease virus. Clones containing cDNA for alpha 2-interferons were identified by colony hybridization with two synthetic hexadecanucleotides. One of the positive clones contained a NH2-terminal part of cDNA of human interferon identical to cDNA for IFN-alpha 2. The only difference between these two clones was the Ser-8 leads to Asn-8 substitution in deduced sequenced of mature interferons. This mutant interferon, named alpha 2, was expressed in E. coli and its properties were compared with those of interferon alpha 2.     

Expression in Escherichia coli of the human leukocyte interferon alpha2 gene fused with N-terminal fragment of beta-galactosidase

Genes for leucocyte interferon and alpha-donor of galactosidase were fused by deletion mutagenesis or by site-directed mutagenesis. In both cases the fused protein was expressed. The protein having an antiviral activity of leucocyte interferon was easily detected in bacteria and solutions by the reaction of beta-galactosidase alpha-complementation and retained the antigenic determinants of interferon and beta-galactosidase. The use of fused proteins for optimization of gene expression and for the analysis of interferon structure-function relationship is discussed.     

Expression of the transglutaminase gene in Escherichia coli.

1. The cDNA gene coding for the enzyme transglutiminase (EC 2.3.2.13) was cloned into the pUC18 oriented for expression from the lac promoter. 2. DNA sequencing of the 5' end showed that the cDNA was missing the sequence coding of the N-terminal 30 amino acids. 3. The truncated gene was then cloned into pKK233-2, and the recombinant product was produced in Escherichia coli. 4. A gene construct coding for the complete protein was generated by inserting an oligonucleotide for the missing 30 amino acids into the Eco RI site of the pUC18 clone. 5. A consensus Shine-Dalgarno sequence and translational start codon were positioned at the 5' end of the linker. 6. Immunoblotting experiments of E. coli JM105(pUC18-TGase) indicated the expression of the transglutaminase gene. 7. The cell lysate as well as the partially purified transglutaminase showed no detectable enzyme activity.     

Overexpression of a synthetic gene encoding human alpha interferon in Escherichia coli

Interferons (IFNs) represent an important defense mechanism in vertebrates. In this work, we describe gene synthesis and assembly using the polymerase chain reaction as a method for single-step synthesis of DNA sequences. The oligonucleotides designed were based on Escherichia coli codon usage and two genes of IFN were synthesized: one containing a DNA sequence already known and the other, a mutated form in which two cysteine amino acid residues were replaced by serines in an attempt to improve the stability of the protein. DNA sequences were cloned into pAE, an E. coli vector that allows heterologous protein expression with or without a histidine tag. Recombinant human interferons (rhIFNs) were identified by Western blotting and ELISA using anti-human interferon polyclonal antibodies. Purification of the recombinant His-tagged proteins was achieved in a single step by Ni(2+)-charged column chromatography while proteins without His-tag were purified by extensively washing the inclusion bodies, the final yields being approximately 210 and 75mg/L, respectively. The rhIFNs expressed within this system were biologically active ( approximately 1,1x10(8)IU/mg) based on antiviral assay. The combined methodologies described here proved to be cost-effective and could be extended to other genes/proteins of interest.     

Expression of the yeast galactokinase gene in Escherichia coli.

In Saccharomyces cerevisiae the genes for three of the enzymes involved in galactose metabolism are tightly linked near the centromere of chromosome II (Douglas and Hawthorne, 1964). However, the molecular mechanisms which control the expression of these genes are not well understood. A DNA fragment containing at least one of these yeast genes, the galactokinase gene (gal1), has been joined to the bacterial plasmid pBR322 and maintained in an Escherichia coli strain that carries a deletion in its own galactokinase gene, galK. The presence of the yeast gene was demonstrated by (i) complementation of the E. coli galactokinase deletion, (ii) by hybridization of the cloned DNA fragment to restriction enzyme digests of total yeast DNA and (iii) by assaying for yeast galactokinase activity in bacterial cell extracts. The yeast DNA fragment is 4700 base pairs long, and enables the host E. coli K-12 strain to grow in minimal medium containing galactose as the sole carbon source with a generation time of 14.3 h. The yeast galactokinase activity in the bacterial extracts is 0.7% of the bacterial galactokinase activity found in wild-type E. coli fully induced with fucose.     

Expression of ovine gamma interferon in Escherichia coli and Corynebacterium glutamicum.

Bacteria of two species, Escherichia coli and Corynebacterium glutamicum, were used as hosts to express recombinant ovine gamma interferon as a fusion protein with glutathione S-transferase. The recombinant gamma interferon produced by both bacteria was biologically active in vitro and was recognized by anti-gamma interferon monoclonal antibodies. E. coli produced large amounts of soluble recombinant protein which could be purified by a simple affinity chromatography method. Only a small fraction of the recombinant protein made by C. glutamicum was recovered by this method. Expression of recombinant protein in C. glutamicum was unstable but could be controlled by increased regulation of the tac promoter. Both hosts expressed ovine gamma interferon at high levels, with the recombinant protein making up a significant proportion of the cellular protein content.     

Expression, refolding, and characterization of human soluble BAFF synthesized in Escherichia coli

The B lymphocyte stimulator (BAFF) is a novel member of the tumor necrosis factor (TNF) ligand family which is important in B lymphocyte maturation and survival. Here, a recombinant form of the extracellular domain of the BAFF (hsBAFF) was expressed in Escherichia coli BL21(DE3) under the control of a T7 promoter. The resulting insoluble bodies were separated from cellular debris by centrifugation and solubilized with 8 M urea. A rapid and simple on-column refolding procedure was developed. It was applied and then the refolded hsBAFF was purified by anion-exchange. The purified final product was >98% pure by SDS-PAGE stained with Coomassie brilliant blue R-250. Mass spectroscopic analysis indicated the protein to be 17.5 kDa, which equalled the theoretically expected mass. The N-terminal sequencing of refolding hsBAFF showed the sequence corresponded to the designed protein. The correct refolding of the recombinant protein was verified in the recovery of its secondary and tertiary structures as assessed by circular dichroism and fluorescence emission spectra. The renatured protein displayed its immunoreactivity with the antibodies to BAFF protein by Western blotting. The final purified material was biologically active in a validated induced human B lymphocyte proliferation bioassay. The expression and in vitro refolding of hsBAFF resulted in production of an active molecule in a yield of 15 mg/L flask cultivation.     

Expression of the x-lor gene of human T-cell leukemia virus I in Escherichia coli.

The 3'-terminal regions of the human T-cell leukemia virus I (HTLV-I) and HTLV-II genomes encode a novel gene product. We showed that expression of this region fused to the beta-galactosidase gene in bacteria produces a protein recognized by adult T-cell leukemia-lymphoma patient sera. Rabbit antibodies raised against this protein specifically precipitated the 42-kilodalton x-lor gene protein from HTLV-I-infected cells.     

Expression of a synthetic gene encoding human transthyretin in Escherichia coli

Transthyretin is an amyloidogenic protein that causes human amyloid polyneuropathy and senile systemic amyloidosis as a result of the deposition of normal and/or mutant transthyretin in the form of amyloid fibrils. A high-expression plasmid of human transthyretin was constructed in order to facilitate the study of amyloid fibril formation of this protein. The transthyretin gene was constructed by an assembly of eight chemically synthesized oligonucleotides and amplified by polymerase chain reaction, and the amplified gene was inserted into an Escherichia coli expression vector. The expression plasmid was transformed into M15 cells and the gene product was expressed as a polyhistidine-tagged fusion protein. Purified recombinant transthyretin was obtained by one-step nickel chelation affinity chromatography and the production level of the protein was 130mg per 1L of culture. Furthermore, the expressed protein showed the same characteristics in terms of tetramer formation at neutral pH and amyloid formation at acidic pH as did the authentic human transthyretin. This system will enable biophysical and structural studies of this protein to be advanced.