Tag removal is a prerequisite issue for structural and functional analysis of affinity-purified membrane proteins. The present study took a MBP-fused membrane protein, MrpF, as a model to investigate the tag removal by TEV protease. Influences of the linking sequence between TEV cleavage site and MrpF on protein expression and predicted secondary structure were investigated. The steric accessibility of TEV protease to cleavage site of MBP-fused MrpF was explored. It was found that reducing the size of hydrophilic group of detergents and/or extending the linking sequence between cleavage site and target protein can significantly improve the accessibility of the cleavage site and promote tag removal by TEV protease.

•New structures in confined space inspired by natural structure-forming processes have been systematically reviewed.•Artificial confinements direct the synthesis of materials with defined structures according to the geometry of confinements.•Biological confinements with specific spatial distribution can control the formation of periodic structure.•Designed confinements with desired modification can provide platforms for the synthesis of three-dimensional nanostructures.•Novel structures will be rationally fabricated in the future with the aid of deeper understanding of biological processes.In living organisms, confined space with specific chemical composition and elaborate spatial distribution regulates the formation of natural structures. Learning from the natural structure-forming process, novel synthesis approaches in deliberated confined systems have been proposed for obtaining designed structures. Artificial confined systems can effectively regulate the synthesis of materials with defined structures according to the geometry of confinements. Collagen fibrils provide biological confinements for the formation of hierarchical structure with periodic arrangement. Genetically engineered living organisms with designed confinements can direct the synthesis of three-dimensional nanostructures. More novel structures will be rationally fabricated in the future with the aid of deeper understanding of biological processes.Download high-res image (341KB)Download full-size image

Confinement is common in biological systems and plays a critical role in the structure-forming process of biominerals. However, the knowledge of confinement effects on biomineralization is limited due to the lack of specific chemical structures and elaborate spatial distribution. In this article, we explore the confined mineralization of amorphous calcium carbonate (ACC) within collagen fibrils. Three issues of the confined mineralization of ACC within collagen fibrils were investigated, including the morphology and characteristics of the confined mineralization of ACC within collagen fibrils; the initiation and development of the confined mineralization of ACC within collagen fibrils; and the driving mechanism of ACC infiltration into collagen fibrils. Results show that the negatively charged ACC droplets were attracted to positively charged gap regions of collagen fibrils through electrostatic interactions, infiltrated into collagen fibrils, and then transformed into the crystalline phase. The observation of juxtaposed crystalline and amorphous phases on the surface of fibrils indicates that a secondary nucleation mechanism may be responsible for the co-orientation of calcite nanocrystals. Through modifying the wettability of amorphous calcium carbonate with magnesium ions, it is verified that the infiltration of ACC into collagen fibrils was driven by capillary forces. The present study not only provides evidence of the confinement effects in biomineralization but also facilitates the understanding of the in vivo bone formation process. It may also open up a new avenue in the bioprocess-inspired synthesis of advanced materials.

TiO2 is a promising anode material for lithium-ion batteries. The electrochemical performance of TiO2 can be improved by optimization of nanostructures. The present study was proposed to control the synthesis of mesoporous nanostructured anatase TiO2 on a genetically modified Escherichia coli surface. A recombinant protein INP-SiliSila containing functional domains of silicatein-α and silaffin was constructed and expressed on the E. coli surface. Deposition of the TiO2 precursor was facilitated by INP-SiliSila on the E. coli surface. Upon calcination, TiO2 coating on the E. coli surface transformed to anatase and formed well-defined rod-shaped particles. The electrochemical performance of the as-prepared anatase TiO2 as anode electrodes was improved and better than that of most reported ones. The present study not only provides an organism-based approach for fabricating nanostructured anatase TiO2 with enhanced electrochemical performance, but also opens a new avenue to take advantage of genetically modified bacterial surfaces in the synthesis and structure control of materials.

Living organisms can produce elegant structures with unique functions and properties through biological processes. Various proteins are involved in these processes. Inspired by the structure formation of mollusc shells, a single multifunctional recombinant protein ChiCaSifi was designed on the basis of mineralization proteins for regulating CaCO3 mineralization in a simple and direct manner. ChiCaSifi contains functional domains of the chitin binding protein (Chi), the calcium binding protein (Ca), and the silk fibroin (Sifi). Therefore, ChiCaSifi can have multiple roles in directing CaCO3 mineralization. Overexpression and purification of ChiCaSifi were achieved. Activities of ChiCaSifi were examined for its binding to calcium and chitin. Influences of ChiCaSifi in regulating the phase formation of CaCO3 crystals on the chitin surface were proved. Structural changes of ChiCaSifi were evidenced and related to its functions on mineralization. These observations indicate that rationally designed proteins with functional domains of mineralization proteins can be effective tools in materials synthesis. The present study may not only provide an insight into the formation of natural biomaterials, but also open a new avenue in the design and synthesis of novel organic–inorganic composite materials.

Taking lessons from the structure-forming process of biominerals in animals and plants, one can find tremendous inspirations and ideas for developing advanced synthesis techniques, which is called bio-process inspired synthesis. Bone, as a typical representative of biominerals, is constituted of mineralized collagen fibrils, which are formed under the functions of non-collagenous proteins (NCPs). Intrafibrillar mineralization is the consequence of a synergy among several NCPs. In the present study, we have designed a multi-functional protein, named (MBP)–BSP–HAP, based on bone sialoprotein (BSP) and hydroxyapatite binding protein (HAP), to mimic the intrafibrillar mineralization process in vitro. The three functional domains of (MBP)–BSP–HAP provide the artificial protein with multiple designated functions for intrafibrillar mineralization including binding calcium ions, binding collagen, and binding hydroxyapatite. Platelet-like hydroxyapatite crystals periodically arranged inside the collagen fibrils have been achieved under the function of (MBP)–BSP–HAP. The mechanism of intrafibrillar mineralization directed by the multi-functional protein was proposed. This work may not only shed light on bio-process inspired approaches for more economic and efficient biomimetic synthesis, but also be helpful in understanding the natural process of bone formation for bone regeneration and tissue repair.

Living organisms can produce elegant structures with unique functions and properties through biological processes. Various proteins are involved in these processes. Inspired by the structure formation of mollusc shells, a single multifunctional recombinant protein ChiCaSifi was designed on the basis of mineralization proteins for regulating CaCO3 mineralization in a simple and direct manner. ChiCaSifi contains functional domains of the chitin binding protein (Chi), the calcium binding protein (Ca), and the silk fibroin (Sifi). Therefore, ChiCaSifi can have multiple roles in directing CaCO3 mineralization. Overexpression and purification of ChiCaSifi were achieved. Activities of ChiCaSifi were examined for its binding to calcium and chitin. Influences of ChiCaSifi in regulating the phase formation of CaCO3 crystals on the chitin surface were proved. Structural changes of ChiCaSifi were evidenced and related to its functions on mineralization. These observations indicate that rationally designed proteins with functional domains of mineralization proteins can be effective tools in materials synthesis. The present study may not only provide an insight into the formation of natural biomaterials, but also open a new avenue in the design and synthesis of novel organic–inorganic composite materials.

Taking lessons from the structure-forming process of biominerals in animals and plants, one can find tremendous inspirations and ideas for developing advanced synthesis techniques, which is called bio-process inspired synthesis. Bone, as a typical representative of biominerals, is constituted of mineralized collagen fibrils, which are formed under the functions of non-collagenous proteins (NCPs). Intrafibrillar mineralization is the consequence of a synergy among several NCPs. In the present study, we have designed a multi-functional protein, named (MBP)–BSP–HAP, based on bone sialoprotein (BSP) and hydroxyapatite binding protein (HAP), to mimic the intrafibrillar mineralization process in vitro. The three functional domains of (MBP)–BSP–HAP provide the artificial protein with multiple designated functions for intrafibrillar mineralization including binding calcium ions, binding collagen, and binding hydroxyapatite. Platelet-like hydroxyapatite crystals periodically arranged inside the collagen fibrils have been achieved under the function of (MBP)–BSP–HAP. The mechanism of intrafibrillar mineralization directed by the multi-functional protein was proposed. This work may not only shed light on bio-process inspired approaches for more economic and efficient biomimetic synthesis, but also be helpful in understanding the natural process of bone formation for bone regeneration and tissue repair.

Confinement is common in biological systems and plays a critical role in the structure-forming process of biominerals. However, the knowledge of confinement effects on biomineralization is limited due to the lack of specific chemical structures and elaborate spatial distribution. In this article, we explore the confined mineralization of amorphous calcium carbonate (ACC) within collagen fibrils. Three issues of the confined mineralization of ACC within collagen fibrils were investigated, including the morphology and characteristics of the confined mineralization of ACC within collagen fibrils; the initiation and development of the confined mineralization of ACC within collagen fibrils; and the driving mechanism of ACC infiltration into collagen fibrils. Results show that the negatively charged ACC droplets were attracted to positively charged gap regions of collagen fibrils through electrostatic interactions, infiltrated into collagen fibrils, and then transformed into the crystalline phase. The observation of juxtaposed crystalline and amorphous phases on the surface of fibrils indicates that a secondary nucleation mechanism may be responsible for the co-orientation of calcite nanocrystals. Through modifying the wettability of amorphous calcium carbonate with magnesium ions, it is verified that the infiltration of ACC into collagen fibrils was driven by capillary forces. The present study not only provides evidence of the confinement effects in biomineralization but also facilitates the understanding of the in vivo bone formation process. It may also open up a new avenue in the bioprocess-inspired synthesis of advanced materials.

Biomineral formation processes in nature are temporally and spatially regulated under the functions of biomolecules in a confined space. It is potentially very productive to rationally design a mineralized system by taking into account confined space as well as biomolecules. The laboratory technique of “bacterial cell surface display” is an ideal platform to host catalytically active proteins in a three-dimensionally confined space. In the present study, aiming to regulate the synthesis of nanostructured TiO2 anatase, repeating segments of silaffin were displayed on Escherichia coli surfaces through genetic manipulation. The displayed protein electrostatically interacted with a titanium source and catalyzed the hydrolysis of titanium dioxide precursors through hydrogen bonding interactions on the cell surface. In the subsequent calcination process, the genetically modified cells not only served as a framework for producing rod-shaped TiO2 assembled by nanoparticles, but also provided a carbon source in situ. The size of nanoparticles was controlled by changing the number of tandem repeats of the protein segment. The as prepared TiO2 anatase exhibited unique characteristics including nanosized anatase crystals, mesoporous structure and carbon coating. When tested as the anode electrode of a lithium-ion battery, it showed excellent lithium storage performance. The carbon coated anatase anode shows a higher specific capacity of 207 mA h g−1 after 200 cycles at a current rate of 1C and an ultra-long cycling lifetime of 5000 cycles with an outstanding retention capacity of 149 mA h g−1 at a higher rate of 10C. This bioprocess-inspired approach may help broaden the scope and impact of nanosized biominerals.