Characterization and purification of polydisperse reconstituted lipoproteins and nanolipoprotein particles

Craig D. Blanchette, Brent W. Segelke, Nicholas O Fischer, Michele H. Corzett, Edward A. Kuhn, Jenny A. Cappuccio, William Henry Benner, Matthew A Coleman, Brett A. Chromy, Graham Bench, Paul D. Hoeprich, Todd A. Sulchek

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Heterogeneity is a fact that plagues the characterization and application of many self-assembled biological constructs. The importance of obtaining particle homogeneity in biological assemblies is a critical goal, as bulk analysis tools often require identical species for reliable interpretation of the results - indeed, important tools of analysis such as x-ray diffraction typically require over 90% purity for effectiveness. This issue bears particular importance in the case of lipoproteins. Lipid-binding proteins known as apolipoproteins can self assemble with liposomes to form reconstituted high density lipoproteins (rHDLs) or nanolipoprotein particles (NLPs) when used for biotechnology applications such as the solubilization of membrane proteins. Typically, the apolipoprotein and phospholipids reactants are self assembled and even with careful assembly protocols the product often contains heterogeneous particles. In fact, size polydispersity in rHDLs and NLPs published in the literature are frequently observed, which may confound the accurate use of analytical methods. In this article, we demonstrate a procedure for producing a pure, monodisperse NLP subpopulation from a polydisperse self-assembly using size exclusion chromatography (SEC) coupled with high resolution particle imaging by atomic force microscopy (AFM). In addition, NLPs have been shown to self assemble both in the presence and absence of detergents such as cholate, yet the effects of cholate on NLP polydispersity and separation has not been systematically examined. Therefore, we examined the separation properties of NLPs assembled in both the absence and presence of cholate using SEC and native gel electrophoresis. From this analysis, NLPs prepared with and without cholate showed particles with well defined diameters spanning a similar size range. However, cholate was shown to have a dramatic affect on NLP separation by SEC and native gel electrophoresis. Furthermore, under conditions where different sized NLPs were not sufficiently separated or purified by SEC, AFM was used to deconvolute the elution pattern of different sized NLPs. From this analysis we were able to purify an NLP subpopulation to 90% size homogeneity by taking extremely fine elutions from the SEC. With this purity, we generate high quality NLP crystals that were over 100 μm in size with little precipitate, which could not be obtained utilizing the traditional size exclusion techniques. This purification procedure and the methods for validation are broadly applicable to other lipoprotein particles.

Original languageEnglish (US)
Pages (from-to)2958-2971
Number of pages14
JournalInternational Journal of Molecular Sciences
Volume10
Issue number7
DOIs
StatePublished - Jul 2009
Externally publishedYes

Fingerprint

Cholates
lipoproteins
Lipoproteins
Size exclusion chromatography
purification
Purification
Gel Chromatography
Apolipoproteins
Atomic Force Microscopy
Polydispersity
HDL Lipoproteins
Electrophoresis
exclusion
Atomic force microscopy
Gels
chromatography
Plague
Liposomes
Phospholipids
Detergents

Keywords

  • Apolipoproteins
  • Atomic force microscopy
  • Bilayer mimetic
  • Lipoprotein crystallization
  • Nanobiotechnology
  • Nanolipoprotein particles
  • Size-exclusion chromatography

ASJC Scopus subject areas

  • Computer Science Applications
  • Molecular Biology
  • Catalysis
  • Inorganic Chemistry
  • Spectroscopy
  • Organic Chemistry
  • Physical and Theoretical Chemistry

Cite this

Characterization and purification of polydisperse reconstituted lipoproteins and nanolipoprotein particles. / Blanchette, Craig D.; Segelke, Brent W.; Fischer, Nicholas O; Corzett, Michele H.; Kuhn, Edward A.; Cappuccio, Jenny A.; Benner, William Henry; Coleman, Matthew A; Chromy, Brett A.; Bench, Graham; Hoeprich, Paul D.; Sulchek, Todd A.

In: International Journal of Molecular Sciences, Vol. 10, No. 7, 07.2009, p. 2958-2971.

Research output: Contribution to journalArticle

Blanchette, CD, Segelke, BW, Fischer, NO, Corzett, MH, Kuhn, EA, Cappuccio, JA, Benner, WH, Coleman, MA, Chromy, BA, Bench, G, Hoeprich, PD & Sulchek, TA 2009, 'Characterization and purification of polydisperse reconstituted lipoproteins and nanolipoprotein particles', International Journal of Molecular Sciences, vol. 10, no. 7, pp. 2958-2971. https://doi.org/10.3390/ijms10072958
Blanchette, Craig D. ; Segelke, Brent W. ; Fischer, Nicholas O ; Corzett, Michele H. ; Kuhn, Edward A. ; Cappuccio, Jenny A. ; Benner, William Henry ; Coleman, Matthew A ; Chromy, Brett A. ; Bench, Graham ; Hoeprich, Paul D. ; Sulchek, Todd A. / Characterization and purification of polydisperse reconstituted lipoproteins and nanolipoprotein particles. In: International Journal of Molecular Sciences. 2009 ; Vol. 10, No. 7. pp. 2958-2971.
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