Cytoplasmic Inclusion Bodies in
            <i>Escherichia coli</i>
            Producing Biosynthetic Human Insulin Proteins

Williams, D.R.G; Frank, RM Van; Wl, Muth; Burnett, J. Paul

doi:10.1126/science.7036343

Cited by 298 publications

(106 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The formation of IBs upon overexpression of heterologous proteins in E. coli is a well-known phenomenon but only recently has it been recognized that it is not due to a passive precipitation of the unfolded protein. 36 Rather, it results from an organized aggregation driven by interactions between hydrophobic stretches of partially folded protein molecules. 37 We hypothesize that in the case of a2N protein, its hydrophobic regions drive the formation of IBs, but it remains partially folded and, therefore, can be efficiently and quantitatively solubilized by FC-12.…”

Section: Discussionmentioning

confidence: 99%

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

et al. 2010

View full text Add to dashboard Cite

V-ATPase is a multisubunit membrane complex that functions as nanomotor coupling ATP hydrolysis with proton translocation across biological membranes. Recently, we uncovered details of the mechanism of interaction between the N-terminal tail of the V-ATPase a2-subunit isoform (a2N ) and ARNO, a GTP/GDP exchange factor for Arf-family small GTPases. Here, we describe the development of two methods for preparation of the a2N 1-402 recombinant protein in milligram quantities sufficient for further biochemical, biophysical, and structural studies. We found two alternative amphiphilic chemicals that were required for protein stability and solubility during purification: (i) non-detergent sulfobetaine NDSB-256 and (ii) zwitterionic detergent FOS-CHOLINEV R 12 (FC-12). Moreover, the other factors including mild alkaline pH, the presence of reducing agents and the absence of salt were beneficial for stabilization and solubilization of the protein. A preparation of a2N 1-402 in NDSB-256 was successfully used in pull-down and BIAcore TM protein-protein interaction experiments with ARNO, whereas the purity and quality of the second preparation in FC-12 was validated by size-exclusion chromatography and CD spectroscopy. Surprisingly, the detergent requirement for stabilization and solubilization of a2N 1-402 and its cosedimentation with liposomes were different from peripheral domains of other transmembrane proteins. Thus, our data suggest that in contrast to current models, so called ''cytosolic'' tail of the a2-subunit might actually be embedded into and/or closely associated with membrane phospholipids even in the absence of any obvious predicted transmembrane segments. We propose that a2N 1-402 should be categorized as an integral monotopic domain of the a2-subunit isoform of the V-ATPase.

show abstract

Section: Discussionmentioning

confidence: 99%

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

et al. 2010

View full text Add to dashboard Cite

show abstract

“…In order to achieve protein stability, PI has been expressed as a fusion protein which is resistant to proteolysis, the N-terminal extension being artificially engineered or selected from bacterial proteins [1][2][3].…”

Section: Resultsmentioning

confidence: 99%

“…As early attempts to obtain recombinant insulin by the expression of the proinsulin gene (coding for the sequence B-chain-C-peptide-A chain) in bacteria failed due to the rapid intracellular degradation of the protein, insulin was attained as a fusion or chimeric protein, carrying at its N-terminus a polypeptide resistant to intracellular degradation [1][2][3]. This sequence is linked to the B-chain N-terminus by a methione residue.…”

Section: Introductionmentioning

confidence: 99%

Expression and folding of an interleukin‐2‐proinsulin fusion protein and its conversion into insulin by a single step enzymatic removal of the C‐peptide and the N‐terminal fused sequence

et al. 1996

View full text Add to dashboard Cite

We report the expression in E. coli of a proinsulin fusion protein carrying a modified interleukin-2 N-terminal peptide linked to the N-terminus of proinsulin by a lysine residue. The key aspects investigated were: (a) the expression of the fused IL2-PI gene, (b) the folding efficiency of the insulin precursor when still carrying the N-fused peptide and (c) the selectivity of the enzymatic cleavage reaction with trypsin in order to remove simultaneously the C-peptide and the N-terminal extension. It was found that this construction expresses the chimeric proinsufin at high level (20%) as inclusion bodies; the fused protein was refolded at 100-200/xg/ml to yield about 80% of correctly folded proinsulin and then it was converted into insulin by prolonged reaction (5 h) with trypsin and carboxypeptidase B at a low enzymelsubstrate rate (I :600). This approach is based on a single enzymatic reaction for the removal of both the N-terminal fused peptide and the C-peptide and avoids the use of toxic cyanogen bromide.

show abstract

“…To generate the appropriate gene fusion, a PvuI/BamHI restriction su(s) fragment including nucleotides (nt) 5832 to 6315 (43) was made blunt ended and ligated into SmaI-linearized pWR590 and pATH10 expression vectors (6,13). The resulting lacZ-and trpE-su(s)648-808 fusion constructs were expressed in E. coli MV1184, and the fusion proteins were partially purified by insoluble aggregation (32,45). The LacZ-Su(s) fusion was further purified by SDS-PAGE on a BioRad Prep-Cell.…”

Section: Methodsmentioning

confidence: 99%

TheDrosophilaSuppressor of sable Protein Binds to RNA and Associates with a Subset of Polytene Chromosome Bands

Murray

Turnage

Williamson

et al. 1997

Molecular and Cellular Biology

View full text Add to dashboard Cite

Mutations of the Drosophila melanogaster suppressor of sable [su(s)] gene, which encodes a 150-kDa nuclear protein [Su(s)], increase the accumulation of specific transcripts in a manner that is not well understood but that appears to involve pre-mRNA processing. Here, we report biochemical analysis of purified, recombinant Su(s) [rSu(s)] expressed in baculovirus and in Escherichia coli as maltose binding protein (MBP) fusions and immunocytochemical analysis of endogenous Su(s). This work has shown that purified, baculovirus-expressed rSu(s) binds to RNA in vitro with a high affinity and limited specificity. Systematic evolution of ligands by exponential enrichment was used to identify preferred RNA targets of rSu(s), and a large proportion of RNAs isolated contain a full or partial match to the consensus sequence UCAGUAGUCU, which was confirmed to be a high-affinity rSu(s) binding site. An MBP-Su(s) fusion protein containing the N-terminal third of Su(s) binds RNAs containing this sequence with a higher specificity than full-length, baculovirus-expressed rSu(s). The consensus sequence resembles both a cryptic 5 splice site and a sequence that is found near the 5 end of some Drosophila transcripts. Immunolocalization studies showed that endogenous Su(s) is distributed in a reticulated pattern in Drosophila embryo and salivary gland nuclei. In salivary gland cells, Su(s) is found both in the nucleoplasm and in association with a subset of polytene chromosome bands. Considering these and previous results, we propose two models to explain how su(s) mutations affect nuclear pre-mRNA processing.The Drosophila melanogaster suppressor of sable [su(s)] gene is one of a group of recessive suppressors that, when mutated, modify phenotypes associated with transposon insertion mutations at particular genes (reviewed in reference 38). Upon further examination at the molecular level, it has become apparent that these suppressor genes encode proteins that function more generally in regulating specific aspects of transcription and pre-mRNA processing. For example, suppressor of white-apricot [su(w a )] encodes an alternative splicing factor (47), and the protein encoded by suppressor of forked [su(f)] is homologous to a human polyadenylation factor subunit (40). The su(s) gene encodes a 150-kDa nuclear protein [Su(s)] with relatively little homology to other known proteins. Voelker et al. (43) identified a region within su(s) encoding 77 amino acids that is 28.6% identical to a region in the Drosophila U1 70K protein (23). This region of U1 70K is thought to be an Arg-Ser domain, which is found in numerous splicing factors (2, 11). Although the corresponding region of Su(s) contains two Argrich clusters, the characteristic Arg-Ser dipeptides are virtually absent from Su(s). Thus, the observed similarity between Su(s) and U1 70K may or may not be functionally significant. Voelker et al. (43) also identified a region of Su(s) with weak homology to an RNA recognition motif (RRM). More recently, this region of su(s) was reexamined, ...

show abstract

Cytoplasmic Inclusion Bodies in Escherichia coli Producing Biosynthetic Human Insulin Proteins

Cited by 298 publications

References 9 publications

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

Expression and folding of an interleukin‐2‐proinsulin fusion protein and its conversion into insulin by a single step enzymatic removal of the C‐peptide and the N‐terminal fused sequence

TheDrosophilaSuppressor of sable Protein Binds to RNA and Associates with a Subset of Polytene Chromosome Bands

Contact Info

Product

Resources

About