With the aim of separating the domains of a bifunctional fusion protein, the ability of several lengths of helix-forming peptides to separate two weakly interacting beta-can domains was compared with that of flexible linkers or of a three alpha-helices bundle domain. We introduced helix-forming peptide linkers A(EAAAK)nA (n = 2-5) between two green fluorescent protein variants, EBFP and EGFP, and investigated their spectral properties. The fluorescence resonance energy transfer from EBFP to EGFP decreased as the length of the linkers increased. The circular dichroism spectra analysis suggested that the linkers form an alpha-helix and the alpha-helical contents increased as the length of the linkers increased. The results clearly suggested the ability of the helical linkers to control the distance and reduce the interference between the domains. This 'linker engineering' may open a way to the rational design of linkers which maximize the multiple functions of fusion proteins or de novo multi-domain proteins.

A new enzymatic protein ligation tool, sortase, has recently emerged from Gram‐positive bacteria. This article outlines the technique, sortase‐mediated ligation, and its applications in protein engineering, which include the introduction of unnatural molecules into proteins, protein immobilization, protein–protein conjugation, protein cyclization, as a self‐cleavable tag for protein expression, protein–PNA hybrids, neoglycoconjugates, and cell‐surface protein labeling, etc.magnified image

Nitrile hydratase from Rhodococcus sp. N-771 is an ab heterodimer with a nonheme ferric iron in the catalytic center. In the catalytic center, aCys112 and aCys114 are modified to a cysteine sulfinic acid~Cys-SO 2 H! and a cysteine sulfenic acid~Cys-SOH!, respectively. To understand the function and the biogenic mechanism of these modified residues, we reconstituted the nitrile hydratase from recombinant unmodified subunits. The ab complex reconstituted under argon exhibited no activity. However, it gradually gained the enzymatic activity through aerobic incubation. ESI-LC0MS analysis showed that the anaerobically reconstituted ab complex did not have the modification of aCys112-SO 2 H and aerobic incubation induced the modification. The activity of the reconstituted ab complex correlated with the amount of aCys112-SO 2 H. Furthermore, ESI-LC0MS analyses of the tryptic digest of the reconstituted complex, removed of ferric iron at low pH and carboxamidomethylated without reduction, suggested that aCys114 is modified to Cys-SOH together with the sulfinic acid modification of aCys112. These results suggest that aCys112 and aCys114 are spontaneously oxidized to Cys-SO 2 H and Cys-SOH, respectively, and aCys112-SO 2 H is responsible for the catalytic activity solely or in combination with aCys114-SOH.

The use of enzymes is a promising approach for site-specific protein modification on living cells owing to their substrate specificity. Herein we describe a general strategy for the site-specific modification of cell surface proteins with synthetic molecules by using Sortase, a transpeptidase from Staphylococcus aureus. The short peptide tag LPETGG is genetically introduced to the C terminus of the target protein, expressed on the cell surface. Subsequent addition of Sortase and an N-terminal triglycine-containing probe results in the site-specific labeling of the tagged protein. We were successful in the C-terminal-specific labeling of osteoclast differentiation factor (ODF) with a biotin- or fluorophore-containing short peptide on the living cell surface. The labeling reaction occurred efficiently in serum-containing medium, as well as serum-free medium or PBS. The labeled products were detected after incubation for 5 min. In addition, site-specific protein-protein conjugation was successfully demonstrated on a living cell surface by the Sortase-catalyzed reaction. This strategy provides a powerful tool for cell biology and cell surface engineering.

The cell membrane is an important interface for communication with extracellular events, and incorporation of bioactive substances, such as antibodies and receptors, into the cell membrane may enhance the potential abilities of cells. Gene manipulation, chemical modification of membrane proteins, and cell surface painting using a GPI anchor have been performed to introduce substances into cell membranes. Furthermore, many lipid anchors have also been used to modify lipid membranes such as liposomes. In this study, we have focused on developing an easy and rapid method for anchoring of substances including macromolecular proteins into the membranes of living mammalian cells. We employed a single oleyl chain derivative coupled with hydrophilic poly(ethylene glycol) (PEG90, the ethyleneoxide (EO) unit is 90) to facilitate solubilization in water. This water-soluble derivative was designated Biocompatible Anchor for Membrane (BAM). Some proteins (streptavidin, EGFP and an antibody) were coupled with BAM90 at the distal terminal of PEG and rapidly (within a few minutes) anchored into the membranes of various cells (NIH3T3, 32D, Ba/F3, hybridoma 9E10). However, the anchored BAM90 disappeared from the cell membranes within 4-5 h in serum-free culture media, and moreover, the retention time of anchoring was shortened (1-2 h) in culture medium containing 10% FBS. We further prepared a dioleylphosphatidylethanolamine (DOPE)-PEG derivative (DOPE-BAM80, the EO unit is 80) as a double oleyl chain derivative for comparison with the single oleyl chain derivative, BAM90. The retention time of anchored DOPE-BAM80 was longer than that of BAM90 and more than 8 h in culture media with and without 10% serum. Furthermore, the treatment time of DOPE-BAM80 for anchoring was nearly as short (within a few minutes) as that of BAM90. In addition, both types of BAMs, BAM90 and DOPE-BAM80, showed no cytotoxicity. Therefore, DOPE-BAM80 is useful for protein anchoring to cells. Although the utilization of BAM90 is considered to be limited, it is expected to useful in restricted environments, for example, in tissues such as the cornea, peritoneum, bladder, and various mucosae, which are less exposed to serum. Thus, we suggest the possibility that both types of BAM can be applied to cell surface engineering.