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Characterization and Purification of Polydisperse Reconstituted Lipoproteins and Nanolipoprotein Particles

Journal Article · · International Journal of Molecular Sciences (Online)
DOI:https://doi.org/10.3390/ijms10072958· OSTI ID:1628357
 [1];  [2];  [2];  [2];  [2];  [2];  [2];  [2];  [2];  [2];  [2];  [3]
  1. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Chemistry, Materials, Earth and Life Sciences; DOE/OSTI
  2. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Chemistry, Materials, Earth and Life Sciences
  3. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Chemistry, Materials, Earth and Life Sciences; Georgia Inst. of Technology, Atlanta, GA (United States). School of Mechanical Engineering
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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.
Research Organization:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division
Grant/Contract Number:
AC52-07NA27344
OSTI ID:
1628357
Journal Information:
International Journal of Molecular Sciences (Online), Journal Name: International Journal of Molecular Sciences (Online) Journal Issue: 7 Vol. 10; ISSN 1422-0067; ISSN IJMCFK
Publisher:
MDPICopyright Statement
Country of Publication:
United States
Language:
English

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Cited By (4)

Imaging and force measurement of LDL and HDL by AFM in air and liquid journal January 2015
Isolation, Characterization, and Stability of Discretely-Sized Nanolipoprotein Particles Assembled with Apolipophorin-III journal July 2010
Cell-Free Co-Translational Approaches for Producing Mammalian Receptors: Expanding the Cell-Free Expression Toolbox Using Nanolipoproteins journal July 2019
Extent of MHC Clustering Regulates Selectivity and Effectiveness of T Cell Responses journal December 2018

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59 BASIC BIOLOGICAL SCIENCES
Biochemistry & Molecular Biology
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apolipoproteins
bilayer mimetic
lipoprotein crystallization
nanobiotechnology
nanolipoprotein particles
size-exclusion chromatography