In Vivo Biotinylation of Bacterial Magnetic Particles by a Truncated Form of Escherichia coli Biotin Ligase and Biotin Acceptor Peptide

Escherichia coli biotin ligase can attach biotin molecules to a lysine residue of biotin acceptor peptide (BAP), and biotinylation of particular BAP-fused proteins in cells was carried out by coexpression of E. coli biotin ligase (in vivo biotinylation). This in vivo biotinylation technology has been applied for protein purification, analysis of protein localization, and protein-protein interaction in eukaryotic cells, while such studies have not been reported in bacterial cells. In this study, in vivo biotinylation of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1 was attempted by heterologous expression of E. coli biotin ligase. To biotinylate BacMPs in vivo, BAP was fused to a BacMP surface protein, Mms13, and E. coli biotin ligase was simultaneously expressed in the truncated form lacking the DNA-binding domain. This truncation-based approach permitted the growth of AMB-1 transformants when biotin ligase was heterologously expressed. In vivo biotinylation of BAP on BacMPs was confirmed using an alkaline phosphatase-conjugated antibiotin antibody. The biotinylated BAP-displaying BacMPs were then exposed to streptavidin by simple mixing. The streptavidin-binding capacity of BacMPs biotinylated in vivo was 35-fold greater than that of BacMPs biotinylated in vitro, where BAP-displaying BacMPs purified from bacterial cells were biotinylated by being mixed with E. coli biotin ligase. This study describes not only a simple method to produce biotinylated nanomagnetic particles but also a possible expansion of in vivo biotinylation technology for bacterial investigation.Biotin/streptavidin binding is the strongest noncovalent interaction known in nature (K_d dissociation constant], ∼10⁻¹⁵ M) (10), and this tight binding is one of the most general tools for biological research and has been widely used for biomolecular detection (11, 12), immobilization (14, 19), and recovery (15). Therefore, it is of great significance to biotinylate biomolecules, in particular, proteins without functional inhibition. For this purpose, the method for site-selective biotinylation of proteins had been developed using biotin ligase. Biotin ligase catalyzes the posttranslational biotinylation of biotin enzymes, such as acetyl coenzyme A (acetyl-CoA) carboxylase, and introduces biotin into a specific lysine residue of a biotin carboxyl carrier protein (BCCP), a subunit of biotin enzymes (13). In early studies, BCCP (∼100 amino acid residues) had been fused with the proteins of interest for biotinylation by biotin ligase (7); however, there was a concern that fused BCCP might disrupt the function of target proteins. Recently, biotin acceptor peptides (BAPs) had replaced BCCP due to the advantage of small size. BAPs, with 15 to 23 amino acid residues, were screened from a peptide library as peptide tags biotinylated by Escherichia coli biotin ligase (4, 25). BAP-fused proteins can be biotinylated outside the cells by adding biotin and purified E. coli biotin ligase with Mg²⁺ and ATP (in vitro biotinylation). Furthermore, it is also possible to biotinylate BAP-fused proteins inside the cells with coexpression of E. coli biotin ligase (in vivo biotinylation) because BAP is specifically recognized only by E. coli biotin ligase. This in vivo biotinylation technology has been applied in eukaryotic cells to purify the proteins by using streptavidin-immobilized resin (8, 24, 28), because biotin/streptavidin interaction permits stringent washing to eliminate the nonspecific binding. Specific biotinylation can be applied also for protein localization analysis. Using fluorophore- or gold nanoparticle-labeled streptavidin, biotinylated proteins were clearly observed in a previous study (27). Recently, a novel technique to detect protein-protein interaction by fusing BAP and biotin ligase was developed by Ting''s group. BAP and biotin ligase were fused to different two proteins, and then the interaction of these proteins was successfully evaluated via biotinylation of BAP (9). In vivo biotinylation technology using heterologously expressed E. coli biotin ligase should be equally useful for prokaryotes; however, such studies have not been reported for bacterial cells.Magnetospirillum magneticum AMB-1, a magnetotactic bacterium, synthesizes intracellular nanosized bacterial magnetic particles (BacMPs) of 50 to 100 nm; these are surrounded by a lipid bilayer membrane, possess a single magnetic domain of magnetite, and exhibit strong ferrimagnetism (18). Furthermore, functional proteins have been displayed on BacMP surfaces through gene fusion techniques (21, 30, 31). BacMP membrane proteins, including Mms13, were used as anchor proteins; this approach permits functional proteins to be localized efficiently and oriented appropriately on BacMPs (31). We recently reported a novel method for the simple production of biotin-labeled magnetic particles through protein display techniques, where introduction of the biotin moiety onto BacMPs was carried out by the endogenous biotin ligase (17). For the biotinylation of BacMPs, we screened the gene encoding BCCP in the AMB-1 genome and displayed it on the surface of BacMPs using an anchor protein, Mms13. BCCP-displaying BacMPs were biotinylated by endogenous AMB-1 biotin ligase in the cells with high efficiency. This in vivo modification approach could be applied for construction of BacMP-quantum dot nanocomposites toward multicolor labeling of cancer cells, where BCCP and antibody carrier protein (protein G) were simultaneously displayed in tandem (16). However, the size of BCCP, with 149 amino acid residues and a mass of 15.6 kDa, makes it rather large for use as a labeling tag. Although it would be preferable to use a smaller peptide, BAP, for the tag to minimize effects on the flanking proteins for future applications, BAP was not recognized and biotinylated by endogenous AMB-1 biotin ligase (17).In this study, in vivo biotinylation of BacMPs was attempted by heterologous expression of E. coli biotin ligase and Mms13-BAP fusion protein in AMB-1 cells. First, the method for effective expression of E. coli biotin ligase in bacterial cells was optimized. Then site-selective biotinylation of BAP on BacMPs was confirmed. Finally, the obvious advantage of in vivo biotinylation of BAP-displaying BacMPs compared with the in vitro biotinylation method was demonstrated.