Properties of soluble fusions between mammalian aspartic proteinases and bacterial maltose-binding protein

Deepali Sachdev, John Chirgwin

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

The mammalian aspartic proteinases procathepsin D and pepsinogen form insoluble inclusion bodies when expressed in bacteria. They become soluble but normative when synthesized as fusions to the carboxy terminus of E. coli maltose-binding protein (MBP). Since these normative states of the two aspartic proteinases showed no tendency to form insoluble aggregates, their biophysical properties were analyzed. The MBP portions were properly folded as shown by binding to amylose, but the aspartic proteinase moieties failed to bind pepstatin and lacked enzymatic activity, indicating that they were not correctly folded. When treated with proteinase K, only the MBP portion of the fusions was resistant to proteolysis. The fusion between MBP and cathepsin D had increased hydrophobic surface exposure compared to the two unfused partners, as determined by bis-ANS binding. Ultracentrifugal sedimentation analysis of MBP-procathepsin D and MBP-pepsinogen revealed species with very large and heterogeneous sedimentation values. Refolding of the fusions from 8 M urea generated proteins no larger than dimers. Refolded MBP-pepsinogen was proteolytically active, while only a few percent of renatured MBP-procathepsin D was obtained. The results suggest that MBP-aspartic proteinase fusions can provide a source of soluble but nonnative folding states of the mammalian polypeptides in the absence of aggregation.

Original languageEnglish (US)
Pages (from-to)127-136
Number of pages10
JournalProtein Journal
Volume18
Issue number1
StatePublished - 1999
Externally publishedYes

Fingerprint

Maltose-Binding Proteins
Aspartic Acid Proteases
Maltose
Bacterial Proteins
Fusion reactions
Pepsinogen A
Sedimentation
Proteolysis
Peptide Hydrolases
Carrier Proteins
Endopeptidase K
Cathepsin D
Amylose
Escherichia coli Proteins
Polypeptides
Inclusion Bodies
Urea
Dimers
Escherichia coli
Bacteria

Keywords

  • Aspartic proteinases
  • Protein folding
  • Protein fusions

ASJC Scopus subject areas

  • Biochemistry
  • Analytical Chemistry
  • Organic Chemistry
  • Bioengineering

Cite this

Properties of soluble fusions between mammalian aspartic proteinases and bacterial maltose-binding protein. / Sachdev, Deepali; Chirgwin, John.

In: Protein Journal, Vol. 18, No. 1, 1999, p. 127-136.

Research output: Contribution to journalArticle

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