CoChat
Sign Up
Cited by 88Protein purification and stabilityLipoproteins and Cardiovascular HealthAlzheimer's disease research and treatments

Human apolipoprotein E expression in Escherichia coli: structural and functional identity of the bacterially produced protein with plasma apolipoprotein E

Open access
Vogel, T, Weisgraber, K H, Zeevi, M I, Ben-Artzi, H, Levanon, A Z, Rall, S C, Innerarity, T L, Hui, D Y +2 more·1985·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.82.24.8696PMC PMC391503
Open this paper in CoChat — free account

Abstract

Human apolipoprotein E (apoE) was produced in Escherichia coli by transforming cells with an expression vector containing a reconstructed apoE cDNA, a lambda PL promoter regulated by the thermolabile cI repressor, and a ribosomal binding site derived from the lambda cII or the E. coli beta-lactamase gene. Transformed cells induced at 42 degrees C for short periods of time (less than 20 min) produced apoE, which accumulated in the cells at levels of approximately equal to 1% of the total soluble cellular protein. Longer induction periods resulted in cell lysis and the proteolytic destruction of apoE. The bacterially produced apoE was purified by heparin-Sepharose affinity chromatography, Sephacryl S-300 gel filtration, and preparative Immobiline isoelectric focusing. The final yield was approximately equal to 20% of the initial apoE present in the cells. Except for an additional methionine at the amino terminus, the bacterially produced apoE was indistinguishable from authentic human plasma apoE as determined by NaDodSO4 and isoelectric focusing gel electrophoresis, amino acid composition of the total protein as well as its cyanogen bromide fragments, and partial amino acid sequence analysis (residues 1-17 and 109-164). Both the bacterially produced and authentic plasma apoE bound similarly to apolipoprotein B,E(low density lipoprotein) receptors of human fibroblasts and to hepatic apoE receptors. Intravenous injection resulted in similar rates of clearance for both the bacterially produced and authentic apoE from rabbit and rat plasma (approximately equal to 50% removed in 20 min). The ability to synthesize a bacterially produced human apolipoprotein with biological properties indistinguishable from those of the native protein will allow the production of large quantities of apoE for use in further investigations of the biological and physiological properties of this apolipoprotein.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1073/pnas.82.24.8696

Cites

40 of 40 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Glycosylation does not affect apoE binding to the LDL receptor.

    “it does not affect apoE binding to the LDL receptor (27, 28)”

    From Preferential association of apolipoprotein E Leiden with very low density lipoproteins of human plasma. (1993) · cites this paper as [27]

    The Results and Discussion report essentially identical LDL-receptor binding for bacterially produced and authentic apoE in fibroblast assays. Our copy does not directly test glycosylation or state that it has no effect on binding, so the claim goes beyond the comparison reported. (LLM, conf=0.78)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Cites

Flagged first, then by citations

Research works printed but not linked: 4

  1. [9]Paik, Y.-K., Chang, D. J., Reardon, C. A., Davies, G. E., Mahley, R. W. & Taylor, J. M. (1985) Proc. Natl. Acad. Sci. USA 82, 3445–3449.
  2. [17]Rall, S. C., Jr., Weisgraber, K. H., Innerarity, T. L. & Mahley, R. W. (1982) Proc. Natl. Acad. Sci. USA 79, 4696-4700.
  3. [27]Kemp, D. J., Coppel, R. L., Cawman, A. F., Saint, R. B., Brown, G. V. & Anders, R. F. (1983) Proc. Natl. Acad. Sci. USA 80, 3787–3791.
  4. [29]Towbin, H., Staehelin, T. & Ġordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354.

Cited by

Sorted by citations

Related

Authors
Vogel, T., Weisgraber, K. H., Zeevi, M. I., Ben-Artzi, H., Levanon, A. Z., Rall, S. C., Innerarity, T. L., Hui, D. Y., Taylor, J. M., & Kanner, D.
Affiliations
University of California, San Francisco (US)
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
1985-12
Volume
82
Issue
24
Pages
8696-8700
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.82.24.8696
PMC
PMC391503
OpenAlex
W2050763556
Cited by
88
References
40
Citations by year
2023: 1 · 2020: 2 · 2019: 1 · 2014: 1 · 2012: 1
FWCI
1.21
Type
journal article
Language
en
Open access
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Health Sciences › Medicine › Surgery › Lipoproteins and Cardiovascular Health
  • Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
Keywords
apolipoprotein E, recombinant protein expression, Escherichia coli expression, cDNA cloning, lambda PL promoter, ribosome binding site, protein purification, plasma clearance, heterologous expression
MeSH terms
Amino Acid Sequence, Apolipoproteins E, Cloning, Molecular, DNA, Escherichia coli, Genetic Vectors, Humans, Isoelectric Point, Molecular Weight, Plasmids, Receptors, LDL, Recombinant Proteins

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review