C-terminal Extension of Truncated Recombinant Proteins in Escherichia coli with a 10Sa RNA Decapeptide
Journal of Biological ChemistryPublished 1 April 1995Open access
Guo-Fen Tu, Gavin E. Reid, Jian‐Guo Zhang, Robert L. Moritz, Richard J. Simpson
Citations234
SJR quartileQ1
SJR score1.71
SNIP1.00
Generate an AI Snapshot to get a quick, structured summary of this paper.
Study Snapshot
ObjectiveStudy objective
MethodsResearch methodology
PopulationPopulation studied
Sample sizeSample sizes
OutcomesStudy outcomes here
ResultsStudy results comes here
LimitationsResearch study limitations comes here
A concise AI-generated summary of the paper will appear here once you click Generate AI Snapshot.
TL;DR
Peptide mapping, electrospray ionization-mass spectrometry, and automated N- and C-terminal sequencing identified a peptide (“tag” peptide), encoded by a small metabolically stable RNA of E. coli attached to truncated C termini of the recombinant protein.
Abstract
When murine interleukin-6 is overexpressed in Escherichia coli, a small population of molecules exhibits a novel C-terminal modification. Peptide mapping, electrospray ionization-mass spectrometry, and automated N- and C-terminal sequencing identified a peptide ("tag" peptide), -Ala-Ala-Asn-Asp-Glu-Asn-Tyr-Ala-Leu-Ala-Ala-COOH, encoded by a small metabolically stable RNA of E. coli (10Sa RNA) attached to truncated C termini of the recombinant protein. A mutant strain of E. coli in which the chromosomal 10Sa RNA gene (ssrA) is disrupted does not produce this C-terminal modification, confirming that the tag peptide originates from the ssrA gene.
Keywords
Biochemistry, Genetics and Molecular BiologyEnvironmental Science
Molecular Cloning: A Laboratory Manual 2
129,982 Citations1989Joseph Sambrook, Elisabeth Fritsch +1 more
The content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories.
GeneThe pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers
6,131 Citations1982Jeffrey Vieira, Joachim Messing
A series of plasmid vectors containing the multiple cloning site (MCS7) of M13mp7 has been constructed and a kanamycin-resistance marker has been inserted into the center of the symmetrical MCS7 to yield a restriction-site-mobilizing element (RSM).
bioRxiv (Cold Spring Harbor Laboratory)A short course in bacterial genetics : a laboratory manual and handbook for Escherichia coli and related bacteria
2,491 Citations1992Jeffrey H Miller
A sequel to Experiments in Molecular Genetics (Cold Spring Harbor Lab), this spiral-bound manual includes 34 detailed experiments with step-by-step protocols and clear diagrams that demonstrate major concep.
Journal of BacteriologyProcessing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure
511 Citations1987Arie Ben‐Bassat, K. L. Bauer +4 more
In vitro analyses with purified enzyme indicated that MAP is a metallo-oligopeptidase with absolute specificity for the amino-terminal methionine, consistent with the deduced specificity of the enzyme based on the analysis of known amino- terminal sequences of intracellular proteins.
Proceedings of the National Academy of SciencesA tRNA-like structure is present in 10Sa RNA, a small stable RNA from Escherichia coli.
417 Citations1994Yuriko Komine, Makoto Kitabatake +3 more
Primer-extension analysis of 10Sa RNA extracted from a bacterial mutant with temperature-sensitive RNase P function revealed that the precursor to 10SaRNA (pre-10Sa RNA) is folded into a pre-tRNA-like structure in vivo such that it can be cleaved byRNase P to generate the 5' end of the mature 10 Sa RNA.
European Journal of ImmunologycDNA cloning of murine interleukin-HP1: homology with human interleukin 6
287 Citations1988Jacques Van Snick, Sylvie Cayphas +5 more
Considering the functional homology that was previously established between these two proteins, HP1 should be renamed murine interleukin 6, and a cDNA sequence isolated from a library prepared using mRNA of a murine helper T cell clone activated with a clonotypic antibody is described.
Journal of Biological ChemistryBiosynthesis and Incorporation into Protein of Norleucine by Escherichia coli
133 Citations1989Gregg Bogosian, Bernard N. Violand +4 more
The methionine analog norleucine was produced during the synthesis of bovine somatotropin by Escherichia coli strain W3110G containing the recombinant plasmid pBGH1 and eliminated by deleting the leucine operon.
BiochemistryPurification of recombinant human tissue factor
109 Citations1989Lisa R. Paborsky, Keri Tate +7 more
The amount of disulfide-linked aggregates is dramatically reduced following immunoaffinity purification of this four-cysteine variant (C2455), which is active in the chromogenic and prothrombin time assays.
Molecular and General Genetics MGGCharacterization of 10S RNA: A new stable RNA molecule from Escherichia coli
84 Citations1979Bimal K. Ray, David Apirion
10S RNA cannot be processed by cell extracts to tRNA-sized molecules and does not bind significantly to ribosomes, hence it is unlikely to be a tRNA precursor or an mRNA.
Molecular and General Genetics MGG10Sa RNA, a small stable RNA of Escherichia coli, is functional
83 Citations1991Bong-Kyeong Oh, David Apirion
A mutant in which the chromosomal 10 Sa RNA gene is interrupted by a cat insert does not produce detectable levels of 10Sa RNA, and it grows more slowly than the parental strain.
Methods in Protein Structure Analysis
80 Citations1995M. Zouhair Atassi, Ettore Appella
New Strategies for Protein and Peptide Characterization: Tracing Cell Signaling Pathways Using a Combination of 2Dgel Electrophoresis and Mass Spectrometry.
European Journal of BiochemistryCharacterisation of a trisulphide derivative of biosynthetic human growth hormone produced in <i>Escherichia coli</i>
79 Citations1994Anne Munk Jespersen, Thorkild Christensen +3 more
A novel protein derivative has been found during process development of biosynthetic human growth hormone; it has been characterised as human growth hormones with a Cys182-Cys189 trisulphide bridge.
European Journal of BiochemistryMurine hybridoma/plasmacytoma growth factor
71 Citations1988Richard J. Simpson, Robert L. MORITZ +2 more
Although there is virtually no similarity between the NH2-terminal region of HP1 and its human biological counterpart (26-kDa protein/interferon-beta 2 = B-cell stimulatory factor-2/interleukin-6), these studies demonstrate extensive amino acid similarity in the middle and COOH-terminAL regions of these molecules suggesting that HP1 is the murine homologue of human interleuk in-6.
Molecular MicrobiologyThe gene for a small stable RNA (10Sa RNA) of <i>Escherichia coli</i>
70 Citations1989Anil K. Chauhan, David Apirion
A gene that codes for a small stable RNA (362 nucleotides) has been sequenced and it is a monocistronic gene, with its own promoter and terminator.
Biochemical and Biophysical Research CommunicationsMistranslation in IGF-1 during over-expression of the protein in Escherichia coli using a synthetic gene containing low frequency codons
68 Citations1988Ramnath Seetharam, Robert A. Heeren +10 more
Partial misincorporation of Lys for Arg has been observed for the Arg residues of IGF-1 when the molecule is expressed in Escherichia coli using a synthetic gene with the low frequency AGA codon encoding all six Arg residues and yeast preferred codons encoding the remaining residues.
Biochemical and Biophysical Research CommunicationsCharacterization of a recombinant murine interleukin-6: Assignment of disulfide bonds
51 Citations1988Richard J. Simpson, Robert L. Moritz +2 more
Murine interleukin 6 (mIL-6) has been synthesized as a fusion protein using a lac operon inducible plasmid in Escherichia coli and it has been shown that there are disulfide bonds between Cys46-Cys52 and Cys75-CYS85.
European Journal of BiochemistryPurification and characterization of a recombinant murine interleukin‐6
48 Citations1992Jian‐Guo Zhang, Robert L. Moritz +3 more
It was demonstrated that complete disulfide bond formation in murine IL-6 occurred during the early urea washing/guanidine hydrochloride extraction steps, so no refolding step was required and, consequently, all were inactive in the hybridoma growth factor assay.
Molecular MicrobiologyZinc and iron regulate translation of the gene encoding <i>Pseudomonas aeruginosa</i> elastase
46 Citations1992Michael J. Brumlik, Douglas G. Storey
The results indicated that zinc regulates the expression of elastase at the translational level and suggest that the iron regulation of lasB occurs predominantly at the transtational level.
Analytical BiochemistryAutomated solid-phase peptide synthesis: Use of 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium tetrafluoroborate for coupling of ert- butyloxycarbonyl amino acids
39 Citations1992Gavin E. Reid, Richard J. Simpson
The use of TBTU in the presence of 1-hydroxybenzotriazole (HOBt) provides a more efficient coupling procedure for Boc-amino acid derivatives, compared to the existing preformed symmetrical anhydride procedure.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular EnzymologyIsolation and characterization of biologically active murine interleukin-1α derived from expression of a synthetic gene in Escherichia coli
27 Citations1989Gaston O. Daumy, Joseph M. Merenda +5 more
The active fragment of mIL-1 alpha appears to consist of aproteinase-sensitive N-terminal region which is not essential for activity, and a proteinase-resistant core which harbors the essential determinants of its cytokine function.
Journal of Microcolumn SeparationsPurification of proteins and peptides for sequence analysis using microcolumn liquid chromatography
21 Citations1992Robert L. Moritz, Richard J. Simpson
Rapid trace enrichment of low nanogram levels of proteins from volumes as high as 500 μL down to 1–2 μL was demonstrated and identification by amino acid sequence analysis of selected peptides recovered from a sub-10 pmol Staphylococcus aureus V8 protease digest of recombinant murine interleukin-6 (IL-6).
FEBS LettersIdentification and characterization of a C‐terminally extended form of recombinant murine IL‐6
12 Citations1991Dennis E. Danley, Christine A. Strick +5 more
The IL‐6 variant with extended C‐terminus was fully active in two separate bioassays, suggesting that the additional amino acids do not disrupt the structure or function of this important region of the molecule.