Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli

Amann, Egon; Brosius, Jürgen; Ptashne, Mark

doi:10.1016/0378-1119(83)90222-6

Cited by 799 publications

(349 citation statements)

References 15 publications

Supporting

Mentioning

345

Contrasting

Unclassified

Order By: Relevance

“…For the production of recombinant human preproinsulin the cDNA was inserted into the newly designed prokaryotic vector pGEX-6T which provides high-level expression of a fusion protein with a histidine hexapeptide and glutathione-S-transferase (GST) at its N-terminus under the control of the tac promoter (Amman et al, 1983).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Recombinant human preproinsulin expression, purification and reaction with insulin autoantibodies in sera from patients with insulin-dependent diabetes mellitus

Berg¹,

Walter

Mauch³

et al. 1993

Journal of Immunological Methods

View full text Add to dashboard Cite

A novel prokaryotic expression vector pGEX-6T was designed for high-level expression of recombinant fusion protein with a histidine-hexapeptide and glutathione-S-transferase at its N-terminus and the recombinant human preproinsulin at its C-terminus. Efficiency of expression was investigated in the Escherichia coli strain CAG456. The synthesized protein was sequestered in an insoluble form in inclusion bodies and was purified to homogeneity by one-step affinity chromatography based on the specific complex formation of the histidine-hexapeptide and a chelating matrix which was charged with Ni 2+ ions. The antigenic nature of the purified recombinant preproinsulin fusion protein was evaluated by ELISA screening for insulin autoantibodies in selected sera from patients with recent-onset type 1 (insulin-dependent) diabetes mellitus classified by the existence of additional autoantibodies reactive against glutamic acid decarboxylase. 14% of the tested sera (n = 43) contained insulin autoantibodies which strongly recognized the recombinant human preproinsulin. Comparable measurements with both recombinant human preproinsulin and mature insulin suggested that the observed autoantigenicity of preproinsulin was mediated by the C-peptide or/and signal peptide.

show abstract

Section: Discussionmentioning

confidence: 99%

“…1), is 5023 bp long and directs the expression of recombinant fusion protein with glutathione-S-transferase (GST) combined with a histidine-hexapeptide as an affinity tail at its Nterminus. The highly efficient synthesis of recombinant protein is controlled by a tac-promoter (Amman et al, 1983) …”

Section: Construction Of Vector Pgex-6tmentioning

confidence: 99%

Recombinant human preproinsulin expression, purification and reaction with insulin autoantibodies in sera from patients with insulin-dependent diabetes mellitus

Berg¹,

Walter

Mauch³

et al. 1993

Journal of Immunological Methods

View full text Add to dashboard Cite

show abstract

“…To obtain optimal regulation of synthesis of the plasmid-encoded ␤ subunit from pKC301 and pKC302, the host strain possesses the compatible pSC101 derivative, pJMH1, which carries lacI q (Thomas & Glass 1991). The multicopy presence of lacI q provides greater repression of the lac promoter than that obtained using lacI q on a single-copy F 0 (our unpublished observations; Amman et al 1983). Expression of ␤ from pKC301 in strain DH5␣/pJMH1 in the presence of 1 mM IPTG (isopropyl-␤-D-thiogalactoside) was estimated to be one to twofold that of the chromosomal level whereas there was no gene product under non-inducing conditions (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Trans‐dominant mutations in the 3′‐terminal region of the rpoB gene define highly conserved, essential residues in the β subunit of RNA polymerase: the GEME motif

Cromie¹,

Ahmad²,

Malik³

et al. 1999

Genes to Cells

View full text Add to dashboard Cite

Background: The multimeric DNA-dependent RNA polymerases are widespread throughout nature. The RNA polymerase of Escherichia coli, which is the most well characterized, consists of a holoenzyme with subunit stoichiometry of ␣ 2 ␤␤ 0 . The ␤ subunit is conserved and has been implicated in all stages of transcription. The extreme C-terminus of the ␤ subunit, which includes two well-conserved sequence segments, contributes to the active centre and has been proposed to act in transcriptional termination. We describe a genetic system for further characterizing the role of the extreme Cterminus of the ␤ subunit of E. coli RNA polymerase. This involves random, PCR (Polymerase Chain Reaction)-mediated mutagenesis of the 3 0 region of rpoB encoding the C-terminal 116 amino acids of ␤, followed by the isolation and characterization of trans-dominant-negative mutations.

show abstract

“…pUC924 contains the 2.6-kb fragment from the BglII site to the third XhoI site, the translational stop codon from ep138 should be used and the resulting non-fusion protein has a size of about 80 kDa. The plasmid pMF924 was constructed from pEA305 (Amann et al, 1983) and the same EglII-XhoI fragment as in pUC924. pEA305 has a tat promoter followed by the N-terminal part of the cI857-coded i, repressor, hence the resulting fusion protein is 17 kDa larger than from pUC924.…”

Section: (C) Computer-predicted Localization Of Antigenic Sitesmentioning

confidence: 99%

“…Yanisch-Perron, et al, 1985) and plasmids pUC8, pUC9, pUC13 (Vieira and Messing, 1982), pEA305 (Amann et al, 1983), pKK240-11 (Amann and Brosius, 1985), pUR278, pUR288 (RUther and MUller-Hill, 1983) and pINIIIAl (Masui et al, 1984) were used.…”

Section: Introductionmentioning

confidence: 99%

Expression of the Epstein-Barr virus 138-kDa early protein in Escherichia coli for the use as antigen in diagnostic tests

Motz

Fan

Seibl

et al. 1986

Gene

View full text Add to dashboard Cite

SUMMARYWe have attempted to produce the 13%kDa early protein (ep138) of Epstein-Barr virus (EBV) in Escherichia coZi. This protein was found, by immunoprecipitation, to be a clinically relevant antigen, especially for the determination of the IgA-titer in patients with nasopharyngeal carcinoma (NPC). Since the expression of the entire ep138 coding region was unsuccessful, we synthesized only the antigenic parts of this protein. Potential antigenic sites were predicted from the amino acid sequence by combining values for hydrophilicity with calculated estimates of the secondary structure. The two predicted fragments were found to be antigenic, but only one of them was stably expressed in E. coli as a non-fusion protein. This stable protein fragment was, in turn, able to stabilize the second antigenic fragment forming an autologous fusion protein, consisting exclusively of EBV-derived sequences. The resulting product reacts particularly well with IgA antibodies of NPC patients indicating its diagnostic value for NPC.

show abstract

Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli

Cited by 799 publications

References 15 publications

Recombinant human preproinsulin expression, purification and reaction with insulin autoantibodies in sera from patients with insulin-dependent diabetes mellitus

Recombinant human preproinsulin expression, purification and reaction with insulin autoantibodies in sera from patients with insulin-dependent diabetes mellitus

Trans‐dominant mutations in the 3′‐terminal region of the rpoB gene define highly conserved, essential residues in the β subunit of RNA polymerase: the GEME motif

Expression of the Epstein-Barr virus 138-kDa early protein in Escherichia coli for the use as antigen in diagnostic tests

Contact Info

Product

Resources

About