Purification of recombinant annexins without the use of phospholipids

Adekunle I. Elegbede, D. K. Srivastava, Anne Hinderliter

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

Due to their involvement in a variety of physiological and pathological processes, different isoforms of annexins are being utilized as markers of some human diseases and bio-imaging of tissue injury (due to apoptosis), and have been proposed as drug delivery vehicles. These, in addition to extensive biophysical studies on the role of annexins in organizing lipid domains in biological membranes, have necessitated development of an efficient protocol for producing annexins in bulk quantities. In this paper, we report a one-step purification protocol for annexin a5 without using lipid vesicles or involving any column chromatographic step. Depending on the growth and expression condition, a fraction of recombinant annexin a5 (cloned in pET3d vector) was sequestered into inclusion bodies. When these inclusion bodies were dissolved in 6 M urea, subjected to a 10-fold snap dilution in the presence of 5 mM Ca2+ and stored overnight at 4 °C, annexin a5 was precipitated as a homogenous protein as judged by SDS-PAGE. This one-step purification protocol produced about 35 mg of highly purified annexin a5 per liter of bacterial culture. The annexin a5 purified from inclusion bodies exhibited similar properties to that obtained from the soluble fraction using the conventional lipid-partitioning approach. Our purification protocol for annexin a5 elaborated herein is equally effective for purification of annexin A2, and we believe, will serve as general protocol for purifying other annexins in bulk quantities for diagnostic as well as detailed biophysical studies.

Original languageEnglish (US)
Pages (from-to)157-162
Number of pages6
JournalProtein Expression and Purification
Volume50
Issue number2
DOIs
StatePublished - Dec 2006
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by National Institutes of Health (NIH) Grants GM 64443 to A.H and CA 113746 to D.K.S.

Keywords

  • Annexin
  • Apoptosis detection
  • Inclusion body
  • Purification
  • Refolding

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