Mass-spectral analysis of human interferon-gamma and chloramphenicol acetyltransferase I produced in two Escherichia coli strains

Recombinant human interferon-gamma and chloramphenicol acetyltransferase I were isolated from two Escherichia coli strains, E. coli LE329 and E. coli XL1-blue and characterized by electrospray ionization mass spectrometry (ESI-MS). The ESI-MS analysis showed higher masses in comparison with the theoretically calculated for both proteins as well as unexpected molecular heterogeneity. The ESI-MS spectral patterns of the proteins depended on the host strain used and were more heterogenous for the proteins isolated from E. coli LE392. One of the proteins (human interferon-gamma obtained from E. coli XL1-blue) was further subjected to BrCN cleavage. The ESI-MS analysis of the polypeptide mixture revealed shift in the molecular mass for two peptides including the last 26 amino acids of the human interferon-gamma molecule.     

Efficiency of a novel non-Shine-Dalgarno and a Shine-Dalgarno consensus sequence to initiate translation in Escherichia coli of genes with different downstream box composition.

The efficiency of a novel non-Shine-Dalgarno translational initiator (ACCUACUCGAGUUAG, denoted PL) to promote translation in Escherichia coli was compared with that of the Shine-Dalgarno (SD) consensus sequence (AAGGAGGU) using four reporter genes. The obtained results showed that the genes of pokeweed antiviral protein (PAP I) and human calcitonin (CT) were poorly expressed under the conventional SD and were better expressed under the PL sequence. On the contrary, the genes of human interferon gamma (hIFN gamma) and chloramphenicol acetyltransferase (CAT) were highly expressed under SD and poorly expressed under the PL sequence. Computer search revealed a great diversity between the four reporter genes in respect to their complementarity to E. coli 16S rRNA. PAP I and CT genes were rich in nucleotides matching 16S rRNA (called downstream boxes) whereas the complementary domains in the other two (hIFN-gamma and CAT) genes were much shorter. The different behavior of the four reporter genes when placed under the translational control of SD and PL sequences was explained by the different binding energy of their mRNAs to the 30S ribosomal subunit.     

Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli

We have previously shown that human interferon α-2b (IFN) produced in Escherichia coli (E. coli) is heterogeneous at the N-terminal, with three major species (Ahsan et al., 2014). These are: (a) the direct translation product of the gene retaining the N-terminal methionine, (b) a species from which the methionyl residue has been removed by E. coli methionyl aminopeptidase to give the native interferon α-2b and (c) in which the N-terminal Cys residue of the latter contains an acetyl group. In this paper we overcome this heterogeneity, using engineered interferon derivatives with phenylalanine residue directly downstream of the N-terminal methionine (Met-Phe-IFN). This modification not only prevented the removal of the N-terminal methionine by E. coli methionyl aminopeptidase but also the subsequent N-acetylation. Critically, Met-Phe-IFN had enhanced activity in a biological assay. N-terminal stabilization was also achieved by fusing human cytochrome b₅ at the N-terminal of interferon (b₅-IFN-chimera). In this case also, the protein was more active than a reciprocal chimera with cytochrome b₅ at the C-terminal of interferon (Met-IFN-b₅-chimera). This latter protein also had a heterogeneous N-terminal but addition of phenylalanine following Met, (Met-Phe-IFN-b₅-chimera), resolved this problem and gave enhanced biological activity.     

Recombinant protein sequences can trigger methylation of N-terminal amino acids in Escherichia coli.

Recombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild-type valine residues at all N-termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHb1.1 was expressed in Escherichia coli, methylation of the N-terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N-terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N-terminal alanine that occurred through two posttranslational events: initial excision of the N-terminal methionine, followed by methylation of alanine as the newly generated N-terminus. No methylation was observed for variants expressed with wild-type valine at the N-terminus of the alpha globin. The methylation of N-terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli. Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N-terminal methylation has been observed previously for native E. coli proteins with similar N-terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli.     

Dr fimbriae coding region associated hemolytic activity of Escherichia coli

Abstract Escherichia coli LE392 (pAL28) was previously isolated as a positive clone harboring the alginate lyase gene ( aly ) from an alginate-degrading strain, Pseudomonas sp. OS-ALG-9. The plasmid pAL205, one of the constructs obtained after successive subcloning of pAL28, gave the highest expression of aly in E. coli cells. A 8-fold increase in the alginate lyase (Aly) activity in E. coli JM109 (pAL205) was induced with isopropyl-β-d-thiogalactoside, which was 210 times higher than that in E. coli LE392 (pAL28). The highly significant increase in the expression of the Aly enzyme with pAL205 was investigated through the nucleotide sequence around the 5' region of aly as well as the N -terminal sequence of the purified enzyme. It was found that the Aly expressed in E. coli (pAL205) was a fused protein containing 7 residues from the N -terminus of β-galactosidase α-peptide and the mature protein found in the Pseudomonas sp. except for three residues in the N -terminal.     

The fate of integrated DNA in Gaeumannomyces graminis transformants

Abstract The interaction of plasminogen with flagella of Escherichia coli was investigated. Plasminogen bound to flagella purified from E. coli LE392, a commonly used cloning host, and E. coli IH3069, and O25H1 strain isolated from a case of newborn bacteremia. The binding was inhibited by the lysine analog ϵ-aminocaproic acid, suggesting involvement of the lysine-binding Kringle domains of plasminogen in the binding. Purified flagella enhanced the formation of plasmin activity in the presence of tissue-type plasminogen activator; a similar enhancement was observed with flagella-expressing LE392 cells.     

Modulation of expression of the human gamma interferon gene in E. coli by site-directed mutagenesis

Plasmids expressing 2 forms of human immune interferon (IFN-gamma) in E. coli have been constructed: 1) pIFNTacI which expresses IFN-gamma with an N-terminal amino acid sequence of met-cys-tyr-cys-gln-, and 2) pIFNTacII which is a derivative of pIFNTacI from which the 9 base pairs (bp) coding for the cys-tyr-cys have been deleted. Quantitation of Western blots showed that approximately 10-fold more IFN-gamma was produced in cells harboring pIFNTacII (7.5% of total cellular protein) as compared to pIFNTacI. The IFN-gamma expressed in E. coli pIFNTacII is biologically active and routinely recoverable at 10(9) units per liter. When examined microscopically, IPTG induced E. coli harboring either plasmid construction contains prominent cytoplasmic inclusion bodies.     

Properties of genetically overproducedE. coli xylose isomerase

Summary Xylose isomerase was purified from a transformedE. coli strain (LE392-pRK248/pTXI-1) (Lastick et al., 1986) that overproduces the enzyme by induction of the strong lambda P_L promotor. Kinetic data, N-terminal sequence analysis, SDS polyacrylamide gel electrophoresis, size exclusion chromatography and immunodiffusion were used to compare the overproduced enzyme with xylose isomerase purified from xylose induced, non-transformedE. coli LE392 cells; no differences between these purified enzyme preparations were found.     

Constitutive expression of a native human interferon-alpha 1 gene in Escherichia coli

1. A plasmid for constitutive expression of the human interferon-alpha 1 (hIFN-alpha 1) gene in Escherichia coli is constructed on the basis of the cloning plasmid pBR322 using a strong synthetic promoter, synthetic ribosome binding site and a native hIFN-alpha 1 gene excised from a chromosomal clone. 2. The yield of recombinant hIFN-alpha 1 from E. coli LE392 cells transformed with the expression plasmid pJP1R9-hIFN-alpha 1 is evaluated to be 2-6 x 10(7) U/l bacterial culture for metabolic shaker and 6-8 x 10(7) U/l for fermentor.     

Immunoprotective extracellular polysaccharides of Klebsiella aerogenes capsular type K1, expressed in Escherichia coli

A gene library of genomic DNA Klebsiella aerogenes of capsular serotype K1 was constructed in E. coli LE392 using the cosmid pMMB33. Culture filtrates of E. coli recombinants were screened by ELISA for extracellular polysaccharides specific for K. aerogenes K1. Extracellular polysaccharide extracts from K. aerogenes K1 and 3% of the E. coli recombinants contained immunoprotective extracellular polysaccharides (IEP) with similar chemical and immunological properties as shown by gel filtration through Sephacryl 1000, double immunodiffusion and mouse protection tests. IEPs contained no detectable protein, had molecular weights of several hundred million and protected mice against lethal autologous K. aerogenes K1 challenge at a dosage of 10 nanograms per mouse.     

Immunoprotective extracellular polysaccharides of Klebsiella aerogenes capsular type K1, expressed in Escherichia coli

Abstract A gene library of genomic DNA Klebsiella aerogenes of capsular serotype K1 was constructed in E. coli LE392 using the cosmid pMMB33. Culture filtrates of E. coli recombinants were screened by ELISA for extracellular polysaccharides specific for K. aerogenes K1. Extracellular polysaccharide extracts from K. aerogenes K1 and 3% of the E. coli recombinants contained immunoprotective extracellular polysaccharides (IEP) with similar chemical and immunological properties as shown by gel filtration through Sephacryl 1000, double immunodiffusion and mouse protection tests. IEPs contained no detectable protein, had molecular weights of several hundred million and protected mice against lethal autologous K. aerogenes K1 challenge at a dosage of 10 nanograms per mouse.     

Direct expression of the extracellular portion of human FcepsilonRIalpha chain as inclusion bodies in Escherichia coli

The extracellular portion of the alpha chain of the human high-affinity IgE receptor (FcepsilonRIalpha) was expressed as inclusion bodies in Escherichia coli. In immunoblot analysis, two bands were reactive to human IgE and mouse anti-human FcepsilonRIalpha monoclonal antibodies. N-terminal sequencing showed that the two bands were equivalent to the soluble FcepsilonRIalpha with a methionine residue at the N-terminus (Met-1-172) and 23-172, in which the N-terminal 22 residues of the soluble FcepsilonRIalpha have been removed, possibly by degradation in E. coli cells. IgE-binding to CHO cells expressing FcepsilonRI was inhibited by the addition of the recombinant products prepared by the refolding procedure from inclusion bodies. The system for the expression of soluble human FcepsilonRIalpha in E. coli presented in this study and its further improvement would be useful for the production of the protein as a potent therapeutic and for analysis of the IgE-FcepsilonRIalpha interaction.     

A simple competitive enzyme-linked immunosorbent assay using antigen-beta-galactosidase fusions

The fusion of the N-terminal 461 bp of the human interferon-alpha 2 (INF) in frame to the beta-galactosidase gene from Escherichia coli is described. The presence of the expected DNA sequence was shown by restriction mapping and DNA sequencing. A fusion protein was demonstrated in crude extracts of E. coli by Western blots using polyclonal anti-beta-galactosidase and monoclonal anti-IFN antibodies. Using monoclonal antibodies specific for the N-terminal region of IFN-alpha and cell-free extracts from an E. coli strain containing the fusion protein, we set up a simple competitive enzyme-linked immunosorbent assay for human interferon. The test described here was linear down to a lower detection limit of at least 1000 Units, or 5 ng human IFN.     

Tandemly repeated hexamer sequences within the beta interferon promoter can function as an inducible regulatory element in activation by the adenovirus E1B 19-kilodalton protein.

The expression of the human beta interferon (IFN-beta) gene was activated by the adenovirus E1B-19K protein. The sequence within the IFN-beta promoter which is related to the activation was analyzed by chloramphenicol acetyltransferase (CAT) assay. The repeated hexamer units, present within the region between -109 and -65 relative to the cap site, were required for the activation of the IFN-beta gene by the E1B-19K protein. The (AAGTGA)8 region, as a typical hexamer of the consensus sequences, was tested for function in the activation by the E1B-19K protein. When the hexamer (AAGTGA)4-8 was inserted upstream of several reporter genes (such as p55cat, pdlE1A-CAT, and pE1B-CAT) which were inefficiently stimulated, the CAT activities of these fusion genes were efficiently stimulated by the E1B-19K protein. These results show that the tandemly repeated hexamer sequences within the IFN-beta promoter can function as an inducible regulatory element in the activation by the adenovirus E1B 19K protein.     

Characterization of human interleukin 2 derived from Escherichia coli.

Interleukin 2 isolated from Escherichia coli cells expressing the human interleukin gene has been characterized. The observed properties of the protein have been compared with those properties which can be deduced from the DNA sequence alone and the published properties of natural human interleukin 2. The purified E. coli-derived interleukin 2 is a monomeric protein of Mr 15 000 with a sedimentation velocity of 1.86S. The amino acid composition of the protein and isoelectric point (7.7) are consistent with that part of the translated DNA sequence of the gene corresponding to the mature protein. A single disulphide bridge was identified between Cys-58 and Cys-105. C.d. suggested that interleukin 2 is predominantly alpha-helical in secondary structure. The E. coli-derived protein differed from natural interleukin 2 in the presence of N-terminal methionine and also in the absence of a carbohydrate moiety. Removal of the coding region for the first three amino acids of the natural interleukin 2 protein sequence (Ala-Pro-Thr) by site-specific mutagenesis resulted in a protein with N-terminal serine. The possibility that the specificity of the E. coli ribosomal methionine aminopeptidase may not recognize the sequence NH2-Met-Xaa-Pro is discussed (where Xaa is any amino acid residue).