Structural schematic of Trx-Balcp19k.

<p>The Trx-Balcp19k is a hybrid protein with C-terminal Balcp19k and N-terminal Trx tag, His tag and S tag, as well as thrombin and enterokinase recognition sites.</p

Adhesion strength of Trx-Balcp19k gel measured using a single-lap-joint shear strength test.

<p>(A) Representative load-distance profile of Trx-Balcp19k gel. (B) Adhesive shear strengths of several protein samples. The aggregated Trx-Balcp19k (Trx-Balcp19k gel) showed a greater adhesion strength (2.10 ± 0.67 MPa) compared to PVA craft glue (1.77 ± 0.54 MPa) and UHU glue (1.78 ± 0.65 MPa), while the disaggregated Trx-Balcp19k (Trx-Balcp19k foam) had an extremely low adhesion strength (40 ± 8 kPa), which was even lower than that of BSA (70 ± 15 kPa).</p

Adhesive aggregation formed by Trx-Balcp19k when dialyzed against Milli-Q water.

<p>(A) Sticky aggregation was generated after Trx-Balcp19k eluate was dialyzed against pure water, which was termed “Trx-Balcp19k gel” for simple description. (B) The slices of lyophilized Trx-Balcp19k gel. (C) The disaggregated Trx-Balcp19k exhibited a foam-like morphology when freeze dried, and therefore it was termed “Trx-Balcp19k foam”.</p

Schematic illustration of the single-lap-joint shear strength test.

<p>Aluminum plate with a thickness of ~1 mm was cut into 100 mm × 10 mm sheets. A small hole was drilled at one end of the aluminum sheet for convenient fixation. During the test, two adhesively-bonded aluminum adherents with an overlapping area of 12 mm × 10 mm were torn apart in the directions indicated by the arrows.</p

Biochemical composition of the Trx-Balcp19k gel.

<p>The SDS-PAGE analysis (A) indicated that the Trx-Balcp19k gel was actually non-covalently aggregated soluble Trx-Balcp19k based on the identical protein bands observed between Trx-Balcp19k gel and Trx-Balcp19k foam. The Trx-Balcp19k gel is proteinaceous, since only trace amounts of carbohydrates (B) and no lipids (C) were detected.</p

Gene cloning results of <i>Balcp19k</i>.

<p>(A) Gel electrophoresis of RT-PCR amplified <i>Balcp19k</i> CDS. Lane M: DL2000 DNA Marker (Takara, Dalian, China), Lane 2: the amplified <i>Balcp19k</i> CDS as indicated by the pentacle. (B) Amino acid sequence alignment of Balcp19k with DNAman 6.0. The Phe⁶⁹ (F) and Ile¹⁰⁶ (I) of AB242295.1 (GenBank ID) were both substituted with Leu (L) in the Balcp19k amplified in this study.</p

A brief comparison of the physical characteristics of the three cp19k homologues.

<p>The molecular weights (MWs) and isoelectric points (pIs) of these three cp19k homologues were predicted with DNAman 6.0 based on their amino acid sequences. The homology was calculated according to the ClustalW2 alignment results of the three cp19k proteins.</p><p>^aThe cp19k homologue in <i>Balanus improvisus</i>.</p><p>A brief comparison of the physical characteristics of the three cp19k homologues.</p

Self-Assembled Nanofibers for Strong Underwater Adhesion: The Trick of Barnacles

Developing adhesives that can function underwater remains a major challenge for bioengineering, yet many marine creatures, exemplified as mussels and barnacles, have evolved their unique proteinaceous adhesives for strong wet adhesion. The mechanisms underlying the strong adhesion of these natural adhesive proteins provide rich information for biomimetic efforts. Here, combining atomic force microscopy (AFM) imaging and force spectroscopy, we examine the effects of pH on the self-assembly and adhesive properties of cp19k, a key barnacle underwater adhesive protein. For the first time, we confirm that the bacterial recombinant <i>Balanus albicostatus</i> cp19k (rBalcp19k), which contains no 3,4-dihydroxyphenylalanine (DOPA) or any other amino acids with post-translational modifications, can self-assemble into aggregated nanofibers at acidic pHs. Under moderately acidic conditions, the adhesion strength of unassembled monomeric rBalcp19k on mica is only slightly lower than that of a commercially available mussel adhesive protein mixture, yet the adhesion ability of rBalcp19k monomers decreases significantly at increased pH. In contrast, upon preassembly at acidic and low-salinity conditions, rBalcp19k nanofibers keep stable in basic and high-salinity seawater and display much stronger adhesion and thus show resistance to its adverse impacts. Besides, we find that the adhesion ability of Balcp19k is not impaired when it is combined with an N-terminal Thioredoxin (Trx) tag, yet whether the self-assembly property will be disrupted is not determined. Collectively, the self-assembly-enhanced adhesion presents a previously unexplored mechanism for the strong wet adhesion of barnacle cement proteins and may lead to the design of barnacle-inspired adhesive materials