Immobilization of histidine-tagged proteins on monodisperse metallochelation liposomes: Preparation and study of their structure.
Liposomes represent a biocompatible platform for the construction of self-assembling proteoliposomes using nickel or zinc metallochelation. Potential applications of such structures consist in the development of new biocompatible vaccination nanoparticles and drug delivery nanoparticle systems. Here, we describe the design and construction of a flow-through ultrafiltration cell suitable for the preparation of monodisperse liposomes enabled for metallochelation and, hence, the formation of proteoliposomes. The linkage of the cell with a fast protein liquid chromatography system facilitates automation of the procedure, which fits the criteria for upscaling. Proof-of-concept experiments are performed using a mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS-NTA-Ni (1,2-dioleoyl-sn-glycero-3-{[N(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl}(nickel salt)) to formulate proteoliposomes with proteins attached by metallochelation, including histidine (His)-tagged recombinant green fluorescent protein and rgp120 (derived from HIV-1 Env). These model proteoliposomes are characterized by gel permeation chromatography and by dynamic light scattering. Transmission electron microscopy and immunogold staining are used to characterize surface-bound proteins, revealing the tendency of rgp120 to form microdomains on liposome surfaces. These microdomains possess a two-dimensional crystal-like structure that is seen more precisely by atomic force microscopy.
Mašek
Josef
J
Department of Pharmacology, Toxicology, and Immunotherapy, Veterinary Research Institute, Brno, Czech Republic.
Bartheldyová
Eliška
E
Korvasová
Zina
Z
Skrabalová
Michaela
M
Koudelka
Stěpán
S
Kulich
Pavel
P
Kratochvílová
Irena
I
Miller
Andrew D
AD
Ledvina
Miroslav
M
Raška
Milan
M
Turánek
Jaroslav
J
D002614:Chelating Agents / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry*; D008823:Micelles; D018625:Microscopy, Atomic Force; D046529:Microscopy, Electron, Transmission; D009532:Nickel / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011510:Proteolipids / Q000737:chemistry; D014462:Ultrafiltration / Q000379:methods
D002614:Chelating Agents; D015699:HIV Envelope Protein gp120; C471213:His-His-His-His-His-His; D055767:Immobilized Proteins; D008081:Liposomes; D008823:Micelles; D009842:Oligopeptides; D011510:Proteolipids; C514793:gp120 protein, Human immunodeficiency virus 1; C020485:proteoliposomes; D049452:Green Fluorescent Proteins; D006639:Histidine; D009532:Nickel