Title: Characterization and Purification of Polydisperse Reconstituted Lipoproteins and Nanolipoprotein Particles

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, yetmore » 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.« less

Authors:

Blanchette, Craig ^[1]; Segelke, Brent ^[1]; Fischer, Nicholas ^[1]; Corzett, Michele ^[1]; Kuhn, Edward ^[1]; Cappuccio, Jenny ^[1]; Benner, William Henry ^[1]; Coleman, Matthew ^[1]; Chromy, Brett ^[1]; Bench, Graham ^[1]; Hoeprich, Paul ^[1]; Sulchek, Todd ^[2]

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+ Show Author Affiliations

1. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Chemistry, Materials, Earth and Life Sciences
2. 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

Publication Date:

Wed Jul 01 00:00:00 EDT 2009

Research Org.:

Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division

OSTI Identifier:

1628357

Grant/Contract Number:  

AC52-07NA27344

Resource Type:

Accepted Manuscript

Journal Name:

International Journal of Molecular Sciences (Online)

Additional Journal Information:

Journal Name: International Journal of Molecular Sciences (Online); Journal Volume: 10; Journal Issue: 7; Journal ID: ISSN 1422-0067

Publisher:

MDPI

Country of Publication:

United States

Language:

English

Subject:

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