Acrylated adhesive proteinic microneedle patch for local drug delivery and stable device implantation.

Microneedle patches have been developed as favorable platforms for delivery systems, such as the locoregional application of therapeutic drugs, and implantation systems, such as electronic devices on visceral tissue surfaces. However, the challenge lies in finding materials that can achieve both biocompatibility and stable fixation on the target tissue. To address this issue, utilizing a biocompatible adhesive biomaterial allows the flat part of the patch to adhere as well, enabling double-sided adhesion for greater versatility. In this work, we propose an adhesive microneedle patch based on mussel adhesive protein (MAP) with enhanced mechanical strength via ultraviolet-induced polyacrylate crosslinking and Coomassie brilliant blue molecules. The strong wet tissue adhesive and biocompatible nature of engineered acrylated-MAP resulted in the development of a versatile wet adhesive microneedle patch system for in vivo usage. In a mouse tumor model, this microneedle patch effectively delivered anticancer drugs while simultaneously sealing the skin wound. Additionally, in an application of rat subcutaneous implantation, an electronic circuit was stably anchored using a double-sided wet adhesive microneedle patch, and its signal location underneath the skin did not change over time. Thus, the proposed acrylated-MAP-based wet adhesive microneedle patch system holds great promise for biomedical applications, paving the way for advancements in drug delivery therapeutics, tissue engineering, and implantable electronic medical devices.

Self-controllable proteinic antibacterial coating with bacteria-triggered antibiotic release for prevention of periprosthetic infection.

Periprosthetic infection is a devastating postimplantation complication in which a biofilm layer harboring invasive microorganisms forms around orthopedic implants, leading to severe implant failure and patient morbidity. Despite the development of several infection-triggered antibiotic release approaches, most current antibacterial coatings are susceptible to undesired antibiotic leakage or mechanical disintegration during prosthesis installation. Herein, we propose a self-controllable proteinic antibacterial coating capable of both long-lasting adherence onto titanium implant substrates over the implant fixation period and instantaneous bacterial eradication. Importantly, the pH-dependent reversible metal coordination of mussel adhesive protein (MAP) enabled bacterial concentration-dependent antibiotic delivery in response to infection-induced acidification. In addition, the MAP coating exhibited superior self-healable adhesive properties and scratch resistance, which enabled to avert issues associated with mechanical damages, including peeling and cracking, often occurring in conventional implant coating systems. The gentamicin-loaded MAP coating exhibited complete inhibition of bacterial growth in vivo against Staphylococcus aureus penetrations during implantation surgery (immediate infection) and even 4 weeks after implantation (delayed infection). Thus, our antibiotic-loaded MAP hydrogel coating can open new avenues for self-defensive antibiotic prophylaxis to achieve instant and sustainable bacteriocidal activity in orthopedic prostheses. © 2017 Elsevier Inc. All rights reserved.

Injectable alginate-based in situ self-healable transparent hydrogel as a vitreous substitute with a tamponading function.

Retinal detachment and other vision-threatening disorders often necessitate vitreous body removal and tamponade injection for retina stabilization. Current clinical tamponades such as silicone oil and expansile gases have drawbacks, including patient discomfort and the need for secondary surgery. We introduce a transparent alginate-phenylboronic acid/polyvinyl alcohol composite hydrogel (TALPPH) as a novel vitreous substitute with tamponading capabilities. In vitro physicochemical, rheological, and optical characterization of in situ self-healable TALPPH was performed, and long-term biocompatibility was assessed in a rabbit model of vitrectomy retinal detachment. In vivo evaluations confirmed TALPPH's ability to inhibit retinal detachment recurrence and preserve rabbit vision without adverse effects. TALPPH's close resemblance to the natural vitreous body suggests potential as a vitreous tamponade substitute for future ophthalmological applications.

Protective Topical Dual-Sided Nanofibrous Hemostatic Dressing Using Mussel and Silk Proteins with Multifunctionality of Hemostasis and Anti-Bacterial Infiltration.

Topical hemostatic agents are preferred for application to sensitive bleeding sites because of their immediate locoregional effects with less tissue damage. However, the majority of commercial hemostatic agents fail to provide stable tissue adhesion to bleeding wounds or act as physical barriers against contaminants. Hence, it has become necessary to investigate biologically favorable materials that can be applied and left within the body post-surgery. In this study, a dual-sided nanofibrous dressing for topical hemostasis is electrospun using a combination of two protein materials: bioengineered mussel adhesive protein (MAP) and silk fibroin (SF). The wound-adhesive inner layer is fabricated using dihydroxyphenylalanine (DOPA)-containing MAP, which promotes blood clotting by aggregation of hemocytes and activation of platelets. The anti-adhesive outer layer is composed of alcohol-treated hydrophobic SF, which has excellent spinnability and mechanical strength for fabrication. Because both proteins are fully biodegradable in vivo and biocompatible, the dressing would be suitable to be left in the body. Through in vivo evaluation using a rat liver damage model, significantly reduced clotting time and blood loss are confirmed, successfully demonstrating that the proposed dual-sided nanofibrous dressing has the right properties and characteristics as a topical hemostatic agent having dual functionality of hemostasis and physical protection.

Hydrogel Microneedles Extracting Exosomes for Early Detection of Colorectal Cancer.

There are several methods for early diagnosis of tumors, such as detecting circulating tumor DNAs, detecting circulating tumor cells, or imaging with tumor-targeting contrast agents. However, these assays are time-consuming and may cause patient discomfort during the biopsy collecting process. Here, we develop a facile method for early diagnosis of tumors by extracting exosomes from interstitial fluid (ISF) using hydrogel microneedles (MNs). The hydrogel MNs expand in the skin to absorb the ISF, and the tumor exosomes contained in the ISF bind with the glypican-1 antibodies inside the hydrogel of MNs. After removing the hydrogel on the MNs, exosomes are separately purified from the ISF to analyze tumor-related biomarkers. Finally, colorectal cancer can be diagnosed by ELISA for the colorectal cancer-induced model mice. This noninvasive hydrogel MN system to obtain the exosome samples would play an important role in early cancer diagnosis.

Tunicate-Inspired Photoactivatable Proteinic Nanobombs for Tumor-Adhesive Multimodal Therapy.

Near-IR (NIR) light-responsive multimodal nanotherapeutics have been proposed to achieve improved therapeutic efficacy and high specificity in cancer therapy. However, their clinical application is still elusive due to poor biometabolization and short retention at the target site. Here, innovative photoactivatable vanadium-doped adhesive proteinic nanoparticles (NPs) capable of allowing biological photoabsorption and NIR-responsive anticancer therapeutic effects to realize trimodal photothermal-gas-chemo-therapy treatments in a highly biocompatible, site-specific manner are proposed. The photoactivatable tumor-adhesive proteinic NPs can enable efficient photothermal conversion via tunicate-inspired catechol-vanadium complexes as well as prolonged tumor retention by virtue of mussel protein-driven distinctive adhesiveness. The incorporation of a thermo-sensitive nitric oxide donor and doxorubicin into the photoactivatable adhesive proteinic NPs leads to synergistic anticancer therapeutic effects as a result of photothermal-triggered "bomb-like" multimodal actions. Thus, this protein-based phototherapeutic tumor-adhesive NPs have great potential as a spatiotemporally controllable therapeutic system to accomplish effective therapeutic implications for the complete ablation of cancer.

Double-layered adhesive microneedle bandage based on biofunctionalized mussel protein for cardiac tissue regeneration.

Heart failure following myocardial infarction (MI), the primary cause of mortality worldwide, is the consequence of cardiomyocyte death or dysfunction. Clinical efforts involving the delivery of growth factors (GFs) and stem cells with the aim of regenerating cardiomyocytes for the recovery of structural and functional integrity have largely failed to deliver, mainly due to short half-lives and rapid clearance in in vivo environments. In this work, we selected and genetically fused four biofunctional peptides possessing angiogenic potential, originating from extracellular matrix proteins and GFs, to bioengineered mussel adhesive protein (MAP). We found that MAPs fused with vascular endothelial growth factor (VEGF)-derived peptide and fibronectin-derived RGD peptide significantly promoted the proliferation and migration of endothelial cells in vitro. Based on these characteristics, we fabricated advanced double-layered adhesive microneedle bandages (DL-AMNBs) consisting of a biofunctional MAP-based root and a regenerated silk fibroin (SF)-based tip, allowing homogeneous distribution of the regenerative factor via swellable microneedles. Our developed DL-AMNB system clearly demonstrated better preservation of cardiac muscle and regenerative effects on heart remodeling in a rat MI model, which might be attributed to the prolonged retention of therapeutic peptides as well as secure adhesion between the patch and host myocardium by MAP-inherent strong underwater adhesiveness.

Sutureless Transplantation of Amniotic Membrane Using a Visible Light-Curable Protein Bioadhesive for Ocular Surface Reconstruction.

The conjunctiva is a thin mucous membrane of the eye. Pterygium, a commonly appearing disease on the ocular surface, requires surgery to excise the conjunctiva to prevent visual deterioration. Recently, transplantation of the amniotic membrane (AM), which is the innermost membrane of the placenta, has been highlighted as an efficient method to cure conjunctiva defects because of its advantages of no side effects compared to mitomycin C treatment and not leaving additional scars on donor site compared to conjunctival autografting. However, to minimize additional damage to the ocular surface by suturing, AM transplantation (AMT) needs to be simplified by using a less invasive, time-saving method. In this work, a visible light-curable protein bioadhesive (named FixLight) for efficient sutureless AMT is applied. FixLight, which is based on bioengineered mussel adhesive protein (MAP), is easily applied between damaged ocular surfaces and transplanted AM, and rapidly cured by harmless blue light activation. Through in vivo evaluation using a rabbit model, the authors demonstrated that FixLight enabled facile, fast, and strong attachment of AM on sclera and promoted ocular surface reconstruction with good biocompatibility. Thus, FixLight can be successfully used as a promising clinical bioadhesive in opthalmological surgeries that require sutureless and rapid operation.

Adhesive protein-based angiogenesis-mimicking spatiotemporal sequential release of angiogenic factors for functional regenerative medicine.

Damaged vascular structures after critical diseases are difficult to completely restore to their original conditions without specific treatments. Thus, therapeutic angiogenesis has been spotlighted as an attractive strategy. However, effective strategies for mimicking angiogenic processes in the body have not yet been developed. In the present work, we developed a bioengineered mussel adhesive protein (MAP)-based novel therapeutic angiogenesis platform capable of spatiotemporally releasing angiogenic growth factors to target disease sites with high viscosity and strong adhesiveness in a mucus-containing environment with curvature. Polycationic MAP formed complex coacervate liquid microdroplets with polyanionic hyaluronic acid and subsequently gelated into microparticles. Platelet-derived growth factor (PDGF), which is a late-phase angiogenic factor, was efficiently encapsulated during the process of coacervate microparticle formation. These PDGF-loaded microparticles were blended with vascular endothelial growth factor (VEGF), which is the initial-phase angiogenic factor, in MAP-based pregel solution and finally crosslinked in situ into a hydrogel at the desired site. The microparticle-based angiogenic-molecule spatiotemporal sequential (MASS) release platform showed good adhesion and underwater durability, and its elasticity was close to that of target tissue. Using two in vivo critical models, i.e., full-thickness excisional wound and myocardial infarction models, the MASS release platform was evaluated for its in vivo feasibility as an angiogenesis-inducing platform and demonstrated effective angiogenesis as well as functional regenerative efficacy. Based on these superior physicochemical characteristics, the developed MASS release platform could be successfully applied in many biomedical practices as a waterproof bioadhesive with the capability for the spatiotemporal delivery of angiogenic molecules in the treatment of ischemic diseases.

Glycan chip based on structure-switchable DNA linker for on-chip biosynthesis of cancer-associated complex glycans.

On-chip glycan biosynthesis is an effective strategy for preparing useful complex glycan sources and for preparing glycan-involved applications simultaneously. However, current methods have some limitations when analyzing biosynthesized glycans and optimizing enzymatic reactions, which could result in undefined glycan structures on a surface, leading to unequal and unreliable results. In this work, a glycan chip is developed by introducing a pH-responsive i-motif DNA linker to control the immobilization and isolation of glycans on chip surfaces in a pH-dependent manner. On-chip enzymatic glycosylations are optimized for uniform biosynthesis of cancer-associated Globo H hexasaccharide and its related complex glycans through stepwise quantitative analyses of isolated products from the surface. Successful interaction analyses of the anti-Globo H antibody and MCF-7 breast cancer cells with on-chip biosynthesized Globo H-related glycans demonstrate the feasibility of the structure-switchable DNA linker-based glycan chip platform for on-chip complex glycan biosynthesis and glycan-involved applications.

Harnessing the bioresponsive adhesion of immuno-bioglue for enhanced local immune checkpoint blockade therapy.

Despite the great promise of immune checkpoint blockade (ICB) therapy for cancer treatment, the currently available options for ICB treatment pose major clinical challenges, including the risk of severe systemic autoimmune responses. Here, we developed a novel localized delivery platform, immuno-bioglue (imuGlue), which is inspired by the intrinsic underwater adhesion properties of marine mussels and can allow the optimal retention of anti-PD-L1 drugs at tumor sites and the on-demand release of drugs in response to the tumor microenvironment. Using a triple-negative breast cancer and melanoma models, we found that imuGlue could significantly enhance anti-tumor efficacy by eliciting a robust T cell-mediated immune response while reducing systemic toxicity by preventing the rapid diffusion of anti-PD-L1 drugs into the systemic circulation and other tissues. It was also demonstrated that imuGlue could be successfully utilized for combination therapy with other immunomodulatory drugs to enhance the anti-tumor efficacy of ICB-based immunotherapy, demonstrating its versatility as a new treatment option for cancer immunotherapy.

Stem cell-loaded adhesive immiscible liquid for regeneration of myocardial infarction.

Myocardial infarction (MI) causes serious loss of cardiac muscle and dysfunction. To restore MI, exogenous stem cells should be efficiently delivered. However, due to severe physical and physiological cardiac environment, recent strategies have faced challenges, including low cell persistence, low integration, and delayed therapeutic effects. Herein, we proposed mesenchymal stem cell (MSC) therapeutic platform using adhesive protein-based immiscible condensed liquid system (APICLS) derived from bioengineered mussel adhesive protein (MAP). With high encapsulation efficiency and survival rate of encapsulated MSCs, APICLS was successfully grafted by intramyocardial injection and distributed throughout the scarred myocardium. Its underwater adhesiveness and biocompatibility fostered integration with damaged tissue, resulting in high cell persistence and maximized paracrine effects. Bioactive molecules released from APICLS with MSCs induced angiogenesis and cardioprotection, delayed cardiac remodeling, reduced fibrosis, and recovered contractive force. Thus, our proposed strategy represents an innovative approach for recovering infarcted cardiac tissues with damaged structural and contractive function.

Coexpression of CMP-sialic acid transporter reduces N-glycolylneuraminic acid levels of recombinant glycoproteins in Chinese hamster ovary cells.

Recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells contain two forms of sialic acids; N-acetylneuraminic acid (Neu5Ac) as a major type and N-glycolylneuraminic acid (Neu5Gc) as a minor type. The Neu5Gc glycan moieties in therapeutic glycoproteins can elicit immune responses because they do not exist in human. In the present work, to reduce Neu5Gc levels of recombinant glycoproteins from CHO cell cultures, we coexpressed cytidine-5'-monophosphate-sialic acid transporter (CMP-SAT) that is an antiporter and transports cytosolic CMP-sialic acids (both forms) into Golgi lumen. When human erythropoietin was used as a target human glycoprotein, coexpression of CMP-SAT resulted in a significant decrease of Neu5Gc level by 41.4% and a notable increase of Neu5Ac level by 21.2%. This result could be reasonably explained by our hypothesis that the turnover rate of Neu5Ac to Neu5Gc catalyzed by CMP-Neu5Ac hydroxylase would be reduced through facilitated transportation of Neu5Ac into Golgi apparatus by coexpression of CMP-SAT. We confirmed the effects of CMP-SAT coexpression on the decrease of Neu5Gc level and the increase of Neu5Ac level using another glycoprotein human DNase I. Therefore, CMP-SAT coexpression might be an effective strategy to reduce the levels of undesired Neu5Gc in recombinant therapeutic glycoproteins from CHO cell cultures.

Multi-dimensional bioinspired tactics using an engineered mussel protein glue-based nanofiber conduit for accelerated functional nerve regeneration.

Limited regenerative capacity of the nervous system makes treating traumatic nerve injuries with conventional polymer-based nerve grafting a challenging task. Consequently, utilizing natural polymers and biomimetic topologies became obvious strategies for nerve conduit designs. As a bioinspired natural polymer from a marine organism, mussel adhesive proteins (MAPs) fused with biofunctional peptides from extracellular matrix (ECM) were engineered for accelerated nerve regeneration by enhancing cell adhesion, proliferation, neural differentiation, and neurite formation. To physically promote contact guidance of neural and Schwann cells and to achieve guided nerve regeneration, MAP was fabricated into an electrospun aligned nanofiber conduit by introducing synthetic polymer poly(lactic-co-glycolic acid) (PLGA) to control solubility and mechanical property. In vitro and in vivo experiments demonstrated that the multi-dimensional tactics of combining adhesiveness from MAP, integrin-mediated interaction from ECM peptides (in particular, IKVAV derived from laminin α1 chain), and contact guidance from aligned nanofibers synergistically accelerated functional nerve regeneration. Thus, MAP-based multi-dimensional approach provides new opportunities for neural regenerative applications including nerve grafting. STATEMENT OF SIGNIFICANCE: Findings in neural regeneration indicate that a bioinspired polymer-based nerve conduit design should harmoniously constitute various factors, such as biocompatibility, neurotrophic molecule, biodegradability, and contact guidance. Here, we engineered three fusion proteins of mussel-derived adhesive protein with ECM-derived biofunctional peptides to simultaneously provide biocompatibility and integrin-based interactions. In addition, a fabrication of robust aligned nanofiber conduits containing the fusion proteins realized suitable biodegradability and contact guidance. Thus, our multi-dimensional strategy on conduit design provided outstanding biocompatibility, biodegradability, integrin-interaction, and contact guidance to achieve an accelerated functional nerve regeneration. We believe that our bioengineered mussel adhesive protein-based multi-dimensional strategy would offer new insights into the design of nerve tissue engineering biomaterials.

Newly Identified HNP-F from Human Neutrophil Peptide-1 Promotes Hemostasis.

Active hemostatic agents can play a crucial role in saving patients' lives during surgery. Active hemostats have several advantages including utilization of natural blood coagulation and biocompatibility. Among them, although human neutrophil peptide-1 (HNP-1) has been previously reported with the hemostatic mechanism, which part of HNP-1 facilitates the hemostatic activity is not known. Here, a partial peptide (HNP-F) promoting hemostasis, originating from HNP-1, has been newly identified by the blood coagulation ability test. HNP-F shows the best hemostatic effect between the anterior half and posterior half of peptides. Moreover, microscopic images show platelet aggregation and an increase in the concentration of platelet factor 4, and the scanning electron microscope image of platelets support platelet activation by HNP-F. Thromboelastography indicates decreased clotting time and increased physical properties of blood clotting. Mouse liver experiments demonstrate improved hemostatic effect by treatment of peptide solution. Cell viability and hemolysis assays confirm the HNP-F's biosafety. It is hypothesized that the surface charge and structure of HNP-F could be favorable to interact with fibrinogen or thrombospondin-1. Collectively, because HNP-F as an active peptide hemostat has many advantages, it could be expected to become a potent hemostatic biomaterial, additive or pharmaceutical candidate for various hemostatic applications.

Marine-derived natural polymer-based bioprinting ink for biocompatible, durable, and controllable 3D constructs.

3D bioprinting (3DBP) is a rapid solid-form fabrication method with a high degree of automation and reproducibility for constructing structural bioscaffolds. However, the development of the 3DBP field has been slowed due to difficulty in acquiring suitable ink materials especially with natural polymers that satisfy all requirements, such as printability, mechanical integrity, and biocompatibility. In this study, a new 3DBP ink of bioengineered sea anemone-derived silk-like protein (aneroin) was used based on its durable mechanical properties and biodegradability in previous studies. The hyaluronic acid and mussel adhesive protein (MAP) were applied for improved printability and cell adhesiveness, respectively. The aneroin-based 3DBP ink (named aneroin ink) was solidified in a few second by dityrosine photo-crosslinking, and its fast reaction was suitable for noncollapsed spaces in printed 3D constructs. Actual-sized human ear, vascular graft, and rectangular multi-layered lattice were bioprinted with high controllability and durable structural integrity. Thus, the developed aneroin ink showed good printability, structural integrity, and biocompatibility for successful application to the construction of various 3D shaped bioscaffolds in tissue and biomedical engineering fields.

Bioinspired Load-Bearing Hydrogel Based on Engineered Sea Anemone Skin-Derived Collagen-Like Protein.

With the help of recombinant DNA technology, many protein candidates have been investigated and engineered for biomaterial applications. Particularly, several repeat sequences with unique secondary structures have been selected as minimal building blocks for biosynthesis to improve the mechanical properties of biomaterials. However, most of these structural proteins have been limited to silk, elastin, collagen, and resilin for decades. In the present work, new repeat sequence found in sea anemone are characterized and biosynthesized into a recombinant protein (named anegen) for potential use as a load-bearing biomaterial. Because its repeat sequence unit has a unique polyproline II structure, which is prevalently found in the triple-helix of collagen, it is assumed to be a promising structural protein candidate that can provide conformational flexibility and elasticity. Because anegen has ≈10% tyrosine residues, inspiration is taken from di-tyrosine crosslinking in the hinge structures of insects, which can be initiated by light activation. It is found that the anegen hydrogel shows higher mechanical properties than a gelatin hydrogel and endures a compression series without deformation. Moreover, the mechanical properties of the anegen hydrogel are controllable through different crosslinking conditions in a wide range of material applications. Importantly, the anegen hydrogel exhibited suitable cell retainability and cell morphology as an implantable biomaterial. Thus, based on its mechanical properties and biocompatibility, the anegen hydrogel can be used as a potential load-bearing and cell-loading scaffolding biomaterial in the tissue and biomedical engineering fields.

Sprayable Adhesive Nanotherapeutics: Mussel-Protein-Based Nanoparticles for Highly Efficient Locoregional Cancer Therapy.

Following surgical resection for primary treatment of solid tumors, systemic chemotherapy is commonly used to eliminate residual cancer cells to prevent tumor recurrence. However, its clinical outcome is often limited due to insufficient local accumulation and the systemic toxicity of anticancer drugs. Here, we propose a sprayable adhesive nanoparticle (NP)-based drug delivery system using a bioengineered mussel adhesive protein (MAP) for effective locoregional cancer therapy. The MAP NPs could be administered to target surfaces in a surface-independent manner through a simple and easy spray process by virtue of their unique adhesion ability and sufficient dispersion property. Doxorubicin (DOX)-loaded MAP NPs (MAP@DOX NPs) exhibited efficient cellular uptake, endolysosomal trafficking, and subsequent low pH microenvironment-induced DOX release in cancer cells. The locally sprayed MAP@DOX NPs showed a significant inhibition of tumor growth in vivo, resulting from the prolonged retention of the MAP@DOX NPs on the tumor surface. Thus, this adhesive MAP NP-based spray therapeutic system provides a promising approach for topical drug delivery in adjuvant cancer therapy.

Versatile signal peptide of Flavobacterium-originated organophosphorus hydrolase for efficient periplasmic translocation of heterologous proteins in Escherichia coli.

Organophosphorus hydrolase (OPH) from Flavobacterium species is a membrane-associated homodimeric metalloenzyme and has its own signal peptide in its N-terminus. We found that OPH was translocated into the periplasmic space when the original signal peptide-containing OPH was expressed in recombinant Escherichia coli even though its translocation efficiency was relatively low. To investigate the usability of this OPH signal peptide for periplasmic expression of heterologous proteins in an E. coli system, we employed green fluorescent protein (GFP) as a cytoplasmic folding reporter and alkaline phosphatase (ALP) as a periplasmic folding reporter. We found that the OPH signal peptide was able to use both twin-arginine translocation (Tat) and general secretory (Sec) machineries by switching translocation pathways according to the nature of target proteins in E. coli. These results might be due to the lack of Sec-avoidance sequence in the c-region and a moderate hydrophobicity of the OPH signal peptide. Interestingly, the OPH signal peptide considerably enhanced the translocation efficiencies for both GFP and ALP compared with commonly used TorA and PelB signal peptides that have Tat and Sec pathway dependences, respectively. Therefore, this OPH signal peptide could be successfully used in recombinant E. coli system for efficient periplasmic production of target protein regardless of the subcellular localization where functional folding of the protein occurs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:848-854, 2016.

Recombinant production of a shell matrix protein in Escherichia coli and its application to the biomimetic synthesis of spherulitic calcite crystals.

OBJECTIVES: To overcome the limited production capability of shell matrix proteins and efficiently conduct in vitro CaCO3 biomineralization studies, a putative recombinant shell matrix protein was prepared and characterized. RESULTS: A glycine-rich protein (GRP_BA) was found in Pinctada fucata as a putative shell matrix protein (NCBI reference sequence; BAA20465). It was genetically redesigned for the production in Escherichia coli. The recombinant protein was obtained in a 400 ml shake-flask culture at approx. 30 mg l(-1) with a purity of >95 %. It efficiently formed a complex with Ca(2+). Ca(2+)-induced agglomeration was like other calcification-related proteins. Spherulitic calcite micro-particles, 20-30 µm diam. with rosette- and sphere-like structures were synthesized in the presence of the recombinant shell protein, which could be formed by stacking and/or aggregation of calcite nanograins and the bound protein. CONCLUSIONS: Recombinant production of a shell matrix protein could overcome potential difficulties associated with the limited amount of protein available for biomineralization studies and provide opportunities to fabricate biominerals in practical aspects.