Efficient and stable display of functional proteins on bacterial magnetic particles using mms13 as a novel anchor molecule

Abstract

Magnetic particles are increasingly used for various biomedical applications because they are easy to handle and separate from biological samples. In this work, a novel anchor molecule was used for targeted protein display onto magnetic nanoparticles. The magnetic bacterium Magnetospirillum magneticum AMB-1 synthesizes intracellular bacterial magnetic particles (BMPs) covered with a lipid bilayer membrane. In our recent research, an integral BMP membrane protein, Mms13, was isolated and used as an anchor molecule to display functional proteins onto BMPs. The anchoring properties of Mms13 were confirmed by luciferase fusion studies. The C terminus of Mms13 was shown to be expressed on the surface of BMPs, and Mms13 was bound to magnetite directly and tightly permitting stable localization of a large protein, luciferase (61 kDa), on BMPs. Consequently, luminescence intensity obtained from BMPs using Mms13 as an anchor molecule was >400 or 1,000 times higher than Mms16 or MagA, which previously were used as anchor molecules. Furthermore, the immunoglobulin G-binding domain of protein A (ZZ) was displayed uniformly on BMPs using Mms13, and antigen was detected by transmission electron microscopy using antibody-labeled gold nanoparticles on a single BMP displaying the ZZ-antibody complex. The results of this study demonstrated the utility of Mms13 as a molecular anchor, which will facilitate the assembly of other functional proteins onto BMPs in the near feature.

FIG. 1.

(A) Amino acid sequence of Mms13. Underlining indicates the putative transmembrane domains, and the arrows indicate fusion site with luciferase. (B) The constructions used are shown. Sequences of thrombin site and luciferase were fused to native Mms13 (Mms13LC) or the first transmembrane domain of Mms13 (dMms13LC). (C) Western blots of proteins in BMP membrane, cytoplasmic, and cell membrane fractions, removed from BMPs of AMB-1 transformants harboring pUMLC. Lane 1, LC (luciferase); 61 kDa, pUM13L; lane 2, Mms13LC (Mms13-luciferase fusion protein; 74 kDa) and pUM13DL; lane 3, dMms13LC (dMms13-luciferase fusion protein; 67 kDa). M, molecular mass marker. BMPs (2.5 mg) or proteins (40 μg) of cytoplasmic and cell membrane fractions were treated with SDS sample buffer. Protein samples were denatured and analyzed by SDS-PAGE and Western blotting using anti-luciferase antibody as described in Materials and Methods.

FIG. 2.

Topology of the C terminus of Mms13 on BMPs identified by proteolytic cleavage. (A) Luminescence intensity of supernatant (100 μl) magnetically separated from BMPs treated without (−) or with (+) thrombin protease (100 U/ml). (B) Western blot of proteins removed from BMPs treated with protease. After being washed with HEPES buffer, BMPs (2.5 mg) were mixed with SDS sample buffer, and the eluted proteins were analyzed by Western blotting as described in Materials and Methods. (C) Schematic diagram showing the reaction of BMPs and thrombin protease.

FIG. 3.

Stability of Mms13-luciferase integrated into the lipid bilayer membrane covering BMPs. (A) Western blot analysis of proteins removed from the lipid bilayer membrane covering BMPs. BMPs (2.5 mg) were mixed with SDS sample buffer, and the eluted proteins were analyzed by Western blotting as described in Materials and Methods. (B) Luminescence intensity of BMPs (20 μg) after washing by pipetting and magnetic separation. (C) SDS-PAGE profile. (D) Western blot analysis of proteins extracted from BMPs with boiling 1% SDS solution. Proteins were extracted from BMPs before (lanes 1) and after (lanes 2) treatment with a solution containing 7 M urea, 2 M thiourea, and 4% CHAPS proteins. Proteins (30 μg) removed from BMPs with boiling 1% SDS solution were used in the experiments shown in panels C and D. Arrowheads indicate Mms13-luciferase.

FIG. 4.

Display of ZZ on BMPs using Mms13. (A) Saturation binding curve of alkaline phosphatase-labeled rabbit anti-goat IgG bound to BMPs. (B) Differential interference microscopy imaging and fluorescence microscopy imaging of BMPs after introduction of TRITC-labeled rabbit anti-goat IgG. WT-BMPs, BMPs extracted from wild-type AMB-1; ZZ-BMPs, BMPs extracted from AMB-1 transformant harboring pUM13ZZ.

FIG. 5.

TEMs of ZZ-BMPs introduced to rabbit IgG after addition of gold nanoparticle (5 nm)-labeled anti-rabbit IgG antibodies (Ab-A) (A) or anti-human IgG antibodies (Ab-B) (B).

FIG. 6.

Observation of sandwich immunoassay on ZZ-BMPs by TEM. (A) TEM of BMPs after sandwich immunoassay. Anti-goat IgG antibody-ZZ-BMP complexes were mixed with antigen (goat IgG, 0.1 to 10,000 ng/ml), followed by gold nanoparticle (10 nm)-labeled anti-goat IgG antibody. (B) Relationship between antigen concentration and number of gold nanoparticles on a single BMP. The number of gold nanoparticles was counted on >50 BMP samples.