Cross-linked protein crystals by glutaraldehyde and their applications

Er-Kai Yan a, Hui-Ling Caoab, Chen-Yan Zhanga, Qin-Qin Lua, Ya-Jing Yea, Jin Hea, Lin-Jun Huanga and Da-Chuan Yin *a
aInstitute for Special Environmental Biophysics, Key Laboratory for Space Bioscience and Space Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, Shaanxi, People's Republic of China. E-mail:; Fax: +86-029-88460254; Tel: +86-029-88460254
bShaanxi Key Laboratory of Ischemic Cardiovascular Disease, Institute of Basic & Translational Medicine, Xi'an Medical University, Xi'an 710021, People's Republic of China

Received 28th January 2015 , Accepted 5th March 2015

First published on 6th March 2015

Cross-linked protein crystal technology, as either a protein stabilisation or enzyme immobilisation method, has garnered more attention recently. This method not only can retain the original activity of the protein molecule but can also significantly enhance the crystals' mechanical and chemical stability. This review presents the preparation and mechanism of cross-linked protein crystals using glutaraldehyde. The mechanical, chemical and thermal properties of the cross-linked protein crystals are also reviewed in detail. In addition, this paper summarises the applications of cross-linked protein crystals in the fields of materials science, biosensors, chromatographic analysis, oral delivery and protein crystal quality improvement. Finally, the limitations and perspectives on cross-linked protein crystals are presented.

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Er-Kai Yan

Er-Kai Yan obtained her bachelor degree in Material Physics at Zhengzhou University of Light Industry (Zhengzhou, China) in 2012. She is now a Ph.D candidate in Materials Science in the School of Life Sciences, Northwestern Polytechnical University. Her research interests include protein crystallogenesis, and cross-linking of protein crystals.

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Da-Chuan Yin

Da-Chuan Yin obtained his Ph.D in Materials Science at Northwestern Polytechnical University (Xi'an, China) in 1996. He became a lecturer at Northwestern Polytechnical University in 1997. In 2006 he obtained a professor position in the School of Life Sciences, Northwestern Polytechnical University. He has started the research in protein crystallization since 2000. Now he is the author of more than 70 scientific research papers and 15 patents. His research interests include protein crystallogenesis, utilization of high magnetic field for materials processing, and biomedical materials for cell culture and tissue engineering.

1 Introduction

Cross-linking is a method that is used to link one polymer to another in both synthetic and natural polymer sciences. The fundamental process for the cross-linking is composed of chemical reactions (with a chemical reagent called as cross-linker) that are usually induced by a change in the environmental parameters, such as temperature, pressure, pH level, etc. The cross-linking of proteins is used in biological sciences to stabilise and study the interactions between protein molecules.

Protein crystals are used predominantly for three-dimensional (3D) structural determination of protein molecules by X-ray diffraction. The importance of the structural information gathered from protein molecules makes it essential to prepare protein crystals. The major characteristic of a crystal is that it consists of a 3D ordered array of protein molecules with solvent (water) and other chemical molecules (small molecules, ions, etc.) filled into the array. This structure leads to relatively weak mechanical properties (i.e., they are soft and fragile), and therefore, the crystal is difficult to handle and use in applications.

A cross-linked protein crystal (CLPC) is a protein crystal treated by the cross-linking process using glutaraldehyde or other bifunctional reagents, and the CLPC possesses significantly different properties from the untreated ones.1 CLPCs are usually stable and insoluble in both water and organic solutions.2 Furthermore, they are typically strong and can be directly handled by mechanical contact. Because of the significant improvement in mechanical properties, CLPCs can be used in applications beyond structural determination by X-ray crystallography, such as nano-materials science, biosensors, chromatographic analyses, and oral delivery. For example, cross-linked enzyme crystals (CLECs) are immobilised for use in enzyme arrays.

Enzymes, which are predominately proteins, are important biological macromolecules that serve as catalysts in many metabolic processes. To make them useful in many applications, one of the major challenges is to improve their stability.3 Several methods have been invented to solve this problem: enzyme immobilisation,4–8 chemical modification,9 protein engineering,10 and medium engineering.11 Among them, enzyme immobilisation is notable for its easier operation and product recovery.12 Additionally, other critical enzyme properties can be improved, such as enzyme purity, stability, activity, specificity, and selectivity.13 In enzyme immobilisation protocols, the chemical cross-linking of enzymes by glutaraldehyde in their crystalline state has attracted considerable attention. Currently, CLECs are potentially useful in many applications, such as the synthesis of pharmaceuticals14 and fine chemicals,15 and the food industry.16

In the field of CLPC technology, there have been well-documented studies and great progress has been made since the middle of last century.17–19 There are many good reviews about the cross-linking of biological macromolecular crystals, such as the papers by Häring et al.,18 Roy et al.,20,21 and Vuolanto.22 Häring et al.18 discussed the application of CLECs in biotransformations and microporous materials; Roy et al.20,21 reviewed the strategies of preparing CLECs and their applications in organic solvent as catalysts; and Vuolanto22 summarised the application of CLPC technology in bioseparation and biocatalysis. Moreover, Zhong23 gave a brief introduction about the stability and production of CLPCs and their applications. Recently, more progress has been made in CLPC technology. The current review aims to provide an up-to-date overview of the properties and applications for CLPCs.

2 Preparation of CLPCs

In 1964, Quiocho et al.24 reported for the first time the treatment of carboxypeptidase-A crystals using a bifunctional reagent (i.e., glutaraldehyde) to obtain CLECs with extensive activity. Generally, the preparation of CLPCs essentially includes two steps: (1) crystallisation of the target proteins and (2) cross-linking the crystals. Fig. 1 illustrates this preparation process for CLPCs.
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Fig. 1 A schematic illustration of the preparation process for CLPCs.

The first step is to obtain protein crystals. Obtaining high quality protein crystals typically requires three steps: (1) protein purification: performed before crystallisation to obtain a protein sample of high enough purity; (2) crystallisation screening: because no one can predict what chemicals will help crystallise a protein that has not been crystallised, many reagents are mixed with the target protein one by one to find the applicable chemicals to effectively crystallise of the protein; and (3) crystallisation optimisation: because the crystals obtained in the crystallisation screening step are most likely of insufficient quality for later use (e.g., in X-ray diffraction), the chemical compositions, pH, temperature, etc. should be adjusted to find the best condition for obtaining crystals with desired quality. Generally, there are four basic methods to achieve protein crystallisation: batch, vapour-diffusion, liquid–liquid diffusion and dialysis. With the development of biological macromolecular crystallisation methodology, the protein crystals can be manipulated into different shapes,25,26 solubilities27 and sizes28 by changing the crystallisation conditions during the crystallising process.

After protein crystals are obtained, the chemical cross-linking of protein crystals can be accomplished using cross-linkers. Several cross-linkers have been used in the cross-linking of protein crystals, and the cross-linkers can be divided into three categories, homobifunctional, heterobifunctional and photoactive cross-linkers. Homobifunctional cross-linkers contain two identical activated reactive groups,29 and their reaction with the protein crystals just needs one step, intra-molecular cross-linking, that can stabilise protein quaternary structure. Heterobifunctional cross-linkers possess two different activated reactive groups,30 and the cross-linking reaction that takes place between the linker and the protein crystals involves two main steps. One terminal reacts with the –NH2 moieties in the protein crystals, and the other terminal typically reacts with the side chains –SH groups. Photoactive cross-linkers are an extension of the heterobifunctional reagents, where one end generally contains the group reacting with the –SH or –NH2 moieties and the other end has the light sensitive groups. Because both terminals are free radicals, the photoactive cross-linkers have high reactivity, but lack specificity. The target functional groups involved in cross-linking reactions include primary amines, mercapto groups, carbonyl groups, hydroxyl groups and carbohydrate chains.

Here, we will briefly introduce several typical cross-linkers, i.e., carbohydrates, thiols, and glutaraldehyde. CLPCs using carbohydrates, particularly glycoproteins, form a Schiff base between the amine groups and the carbohydrate moieties.21 Margolin et al.31 described carbohydrate cross-linked crystals of glucose oxidase, Candida rugosa lipase and several vaccine antigens. Cross-linking using thiols forms heteroconjugates, which occur between the amine and the thiol groups.21 Cross-linking using glutaraldehyde forms strong covalent bonds between the amino groups and the aldehyde groups.9 Roy et al.32 and Rajan et al.33 prepared CLECs of laccase and lipase, respectively, using glutaraldehyde as the cross-linker. Among the cross-linkers listed, glutaraldehyde acts as an organic cross-linker and is one of the most effective protein cross-linkers based on its unique properties, such as high efficiency, low toxicity, high binding capacity, and easy operation.

The cross-linking reaction for protein crystals can be optimised by varying the cross-linking conditions, such as pH, temperature, reaction time, and glutaraldehyde concentration. Lusty34 introduced a gentle method, where glutaraldehyde was introduced through vapour diffusion into the crystallisation solution and then used to cross-link the protein crystals. CLPCs obtained by this approach were not only more resistant to the lattice disorder induced by the cryocooling used for X-ray diffraction but also more stable in solutions different from the mother liquor.35 Iimura et al.36 conducted an epitaxial growth assay on the reusable seed crystal. Protein crystals were cross-linked by glutaraldehyde in advance, and the layer that grew on the seed crystal possessed similar lattice constants, crystallographic direction and surface morphology to those of the as-grown crystal. Large crystals up to 5 mm can be obtained through this technique. Quiocho et al.37 successfully prepared cross-linked carboxypeptidase-A crystals using three distinctive cross-linkers, showing that one type of protein crystal can be cross-linked by multiple different cross-linkers.

3 Mechanism of CLPCs formation using glutaraldehyde

As a versatile and highly efficient cross-linker, glutaraldehyde has already been widely used for the stabilisation of protein crystals. However, the cross-linking mechanism is still not fully clarified38 due to the complexity of the reactions during the cross-linking process.

Glutaraldehyde is a straight-chain saturated dialdehyde with 5 carbons. It is soluble in water or organic solvents such as ethanol and chloroform. The behaviour of glutaraldehyde in aqueous solution has been extensively investigated.38–40 In aqueous media, the two free aldehyde groups in the glutaraldehyde molecules are very reactive; thus, there is strong capacity for self-polymerisation38 (Fig. 2). The pH has a profound effect on the polymerisation rate of glutaraldehyde in solution.41 In acidic conditions, glutaraldehyde exhibits stable performance and low polymerisation.42 In alkaline conditions, however, it undergoes polymerisation,39,40 yielding a mixture of unsaturated polymers of different structures and lengths.43,44 The concentration of glutaraldehyde is also important. With increasing glutaraldehyde concentration, the polymerisation rate increases.

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Fig. 2 A schematic diagram of the polymerisation of glutaraldehyde.21

In 1977, Yonath et al.45 investigated triclinic lysozyme crystals cross-linked with glutaraldehyde and determined that the cross-linking reaction of glutaraldehyde was with lysine residues on the crystal surface using an electron density difference maps. The predominant cross-linking mechanism of glutaraldehyde in protein crystals is the reaction of the aldehyde groups with the ε-amino groups of lysine residues1,46 (Fig. 3), which ensures that the protein crystals are stable and of highly active. Persichetti et al.47 showed that part of lysine ε-amino groups in thermolysin CLECs were accessible to aldehyde molecules, and the other lysine residues were not modified. Apart from the aforementioned dominant reaction, reactions between glutaraldehyde (or its polymerisation products) and different enzyme moieties, such as amines, thiols, and imidazoles, are also possible.46 Therefore, in the cross-linking process, two reactions occur: the polymerisation of glutaraldehyde as well as its (or the polymerised forms') chemical cross-linking with different amino groups; therefore, the reaction mechanism of glutaraldehyde with proteins is not limited to just one mechanism. Cross-linking reactions may occur between different protein molecules (intermolecular cross-linking) or between groups in the same molecule (intramolecular cross-linking),48,49 which make the cross-linking process even more complex. The crystal structure, polymerisation of glutaraldehyde, pH, and temperature are all important factors in preparing cross-linked protein crystals successfully.

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Fig. 3 Schematic representation of the chemical cross-linking between amino and aldehyde groups.21

Though the process is undoubtedly complicated, Wine et al.50 showed that the reaction to cross-link protein crystals using glutaraldehyde was not random. There was a specific initiation site more accessible to cross-linking in the neighbouring protein molecule, and the reaction rate was much slower under acidic conditions compared to alkaline conditions. Furthermore, the cross-linking reactions corresponding to different reaction mechanisms were derived from different trigger points and reaction products in acidic and alkaline pH. Because there are solvent channels throughout the entire protein crystal, chemical cross-linking between amino and aldehyde groups can proceed to completion.20 However, because the external molecules are more accessible to the aldehyde moieties than the internal molecules, the cross-linking reaction may be inhomogeneous and the CLPCs may crack from the internal stress arising from inhomogeneous heat-expansion and cold-contraction.

4 Properties of CLPCs

4.1 High purity

Crystallisation is a well-known approach to obtain highly purified proteins. During crystallisation, protein molecules can be extracted and packed into a three-dimensionally ordered array from a mixture containing mumerous impurities. Recrystallisation can further enhance the purity. During the cross-linking process, few impurities are introduced; hence, CLPCs are usually highly pure.

4.2 High stability

Untreated protein crystals are typically only stable in the solution from which they are grown. When these crystals are placed into other environments, they will in most cases lose their three-dimensional lattice structure. Such unstable behaviour certainly limits their applications. Compared with the corresponding soluble protein crystals, the chemical cross-linking of protein crystals via glutaraldehyde can exceptionally enhance their stability against denaturation, aggregation or decomposition in harsh circumstances.51,52 Moreover, it permits broader options for reaction conditions and allows the use of high concentrations of organic compounds. As a result, the mechanical properties19,53,54 and thermal stability25,55 of protein crystals will be improved.

The mechanical properties are enhanced through cross-linking reactions. Margolin et al.53 reviewed the mechanical properties of cross-linked lysozyme crystals. The results indicated that chemical cross-linking enhanced the mechanical stability of protein crystals, and the improvement depended on the crystal form. Lee56 showed that the mechanical properties of the CLECs of yeast alcohol dehydrogenase I were highly related to the size and crystal from. Lee et al.57 compared the mechanical stability of two lattice structure of cross-linked alcohol dehydrogenase crystals at 27[thin space (1/6-em)]000 rpm. They found that abrasion occurred in hexagonal crystals, while the rod-like crystals did not exhibit breakage in the same conditions, indicating that the mechanical stabilisation of CLPCs was related to the crystal form. These results corroborated previous research by both Margolin and Lee.

Thermal stability is improved via chemical cross-linking by glutaraldehyde. Noritomi et al.55 found that the thermal stability of cross-linked subtilisin crystals in an organic solvent was significantly improved compared with free subtilisin. Furthermore, the half-life of the cross-linked subtilisin crystals was 200 days in octane, whereas the half-life of the free subtilisin crystals was only 5.4 days under the same conditions. In addition, the stability of the cross-linked subtilisin crystals in octane was much higher than that in acetonitrile. Ayala et al.58 found that the thermal stability of cross-linked chloroperoxidase crystals was significantly improved compared to the free chloride oxide enzyme.

The improved stability of CLPCs may stem from the fact crystallized protein forms a lattice and that the protein molecules are covalently cross-linked.59 When protein transitions from a free state to a crystalline state, the protein molecules change from disordered to ordered and become arranged in a regular three-dimensional structure. The concentration of the protein molecules in the crystalline state is close to the theoretical limit, leading to massive protein–protein molecular interactions, such as static electricity and hydrophobic interactions.60 The bonding interactions between molecules play a powerful role in improving the stability of CLPCs. In addition, there are intermolecular cross-links in the crystals after chemical cross-linking by glutaraldehyde. Furthermore, the resistance of CLPCs to proteolysis is significantly enhanced.17 When compared with soluble protein, the stability (resistance to pH, temperature, organic solvent, proteolytic enzymes, etc.) of the cross-linked protein crystals increases significantly, and the degree of stability improvement depends on the crystalline lattice, the static electricity, the hydrophobic interactions and the covalent cross-linking between glutaraldehyde and protein molecules.

4.3 High activity in organic solvent

As high-performance biocatalysts, enzymes are required to maintain their stable structure and high activity during their reaction, particularly in organic systems.61–63 In fact, the catalytic activity of an enzyme in organic media is closely related to the pH, water content, and size and shape of the enzyme.64,65 Unlike water molecules, organic solvents lack the ability to accommodate hydrogen bonds and also exhibit low dielectric constants; thus, enzymatic activity in organic solvents is lower than in water.66 Different organic solvents show different influences on catalysis. Broadly speaking, the nature of the solution, such as the hydrophobicity, the ability to organise hydrogen bonding and the water miscibility, has a great impact on the structure and the catalytic activity of an enzyme.

The low activity of enzymes in organic solvents has limited their application in the field of synthesis.67 Khalaf et al.2 presented a detailed study on the catalytic activity of CLECs in organic solvents, demonstrating for the first time that the activity of pure lipase did not decrease in an organic solvent, and they also discovered that cross-linked wrinkled Candida lipase crystals showed higher enantioselectivity than the crude purified Candida lipase. Thus, CLECs were proposed as active biocatalysts17,68 that could not only withstand certain shear stress, but also be stable in water and organic solvents.69,70 Noritomi et al.71 discovered that the CLECs of subtilisin exhibited higher activity and enantioselectivity than the free state in an organic solvent, and the activity in decane was 780 times higher than that in triethylamine. In fact, Roy et al.32 reported that cross-linked enzyme crystals of laccase had a higher activity in non-polar organic solvents, such as hexane, toluene, isooctane and cyclohexane. Moreover, protein crystals cross-linked by glutaraldehyde or another bifunctional reagent could also increase resilience.35 The lipase CLEC had improved thermal properties and reuse robustness, exhibiting good activity in both polar and nonpolar organic solvents.33 Roy et al.72 showed that the laccase CLEC maintained its high performance after reuse for the biotransformation of pyrogallol to purpurogallin, and the reaction rate did not decrease even after 650 h of continuous utility. CLECs preserve catalytic activity under detrimental conditions such as elevated temperature, extreme pH, and even in the presence of radical chemicals.47,73,74

5 Applications of CLPCs

After extensive investigation, the CLPCs now have potential applications in nano-materials preparation, biosensors, chromatographic analysis, oral delivery and crystal quality improvement.

5.1 Preparation of nano-materials

5.1.1 Preparation of inorganic nanoparticles. The structure of protein crystals is rather porous and nano-fibrous. Solvent channels in protein crystals are typically in the range of 0.5–10 nm in diameter and the volume of these channels (or the pores) in the crystals is approximately 30–65% of the total crystal volume.75 This structure makes the crystals ideal passages for mass transfer and perfect templates for nano-material deposition in chemical reactions. When protein crystals are cross-linked, the preparation of nano-materials using CLPCs as templates becomes possible, because the cross-linked crystals not only retain their biological activity but also enhance their mechanical and chemical properties so that they are very stable during the preparation of nano-materials.

Nano-materials can be fabricated by soaking the CLPCs in an ionic solution, followed by an oxidation–reduction reaction in the solvent channel to obtain molecules that are finally deposited in the channel and become nano-structured materials (Fig. 4). These materials possess the advantages of both bio-molecules and nano-materials, thus they show very important optical, magnetic, electronic, chemical and biological properties. Recently, the nano-materials synthesised by CLPCs are mainly inorganic and organic polymer nano-structured materials.

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Fig. 4 Schematic diagram of nano-particles preparation using CLPCs as biotemplates. Metal nano-particles. Elemental metal nano-particles are one of the simplest structures and are often referred to as particles of gold, silver, platinum and other precious metals.76 Metal nano-particles have attracted considerable attention for their unique physicochemical properties, such as high hardness, excellent conductivity and large surface area.77

Recently, numerous studies have been carried out on the preparation of nano-particles using CLPCs as templates. Falkner et al.78 immersed porous cross-linked crystals of cowpea mosaic virus into potassium tetrachloroplatinate(II) hydrate and sodium hypophosphite buffer. The Pt2+ was reduced by hypophosphite and deposited in the solvent channel; thus, platinum nano-particles were successfully prepared. Guli et al.79 used cross-linked lysozyme crystals as templates to grow periodically arranged Ag/Au nanostructures in AgNO3 and AuCl4 solutions through chemical reduction or UV light treatment. Subsequently, Muskens et al.80 presented extensive studies on the optical response of Ag and Au infiltrated CLLCs by angle- and polarisation-dependent spectroscopy. In an example of template engineering, Wei et al.81 synthesised gold nano-particles within single lysozyme crystal without destroying the native protein structure using an in situ approach. Following their preparation of gold nano-particles using native protein crystals, Wei et al.82 further studied the activity of the gold nano-particles within the single lysozyme crystal (AuNPs@Lys) in the catalytic reduction of p-nitrophenol to p-aminophenol by NaBH4. The results indicated that AuNPs@Lys could be successfully used as biocatalysts with superior performance. Recently, several studies by Miao Liang et al.83,84 have shown that an array of densely packed silver/gold nano-particles can be formed using CLECs as bio-templates for chemical reduction without chemical or physical treatments. Furthermore, these researchers also demonstrated that the AuNPs@CLLCs and AgNPs@CLLCs can be used as recyclable catalysts in the reduction of 4-nitrophenol to 4-aminophenol. Nano-particles of inorganic compounds. Compared with metal nano-particles, nano-materials of inorganic compounds are more complex in their structure and composition. Recently, there several novel nano-particles have been prepared, such as quantum dots,85 magnetic nano-particles,86 etc. CLPCs can be used in preparation of these nano-particles. For examples, Abe et al.87 prepared bimetallic CoPt nano-particles with different magnetic properties in the inner surface of the CLLC pores by soaking, washing, chemically reducing of Co2+ and Pt2+, along with other treatments. Wei et al.88 described the growth of CdS quantum dots within intact single lysozyme crystals. This nano-material-in-crystal hybrid transmitted stronger red fluorescence than the substance without the crystal, and adding Ag(I) or Hg(II) changed the fluorescence properties. Organic nano-particles. The combination of polymers and nano-particles leads to the formation of organic polymer nano-particles that possess favourable storage stability and biocompatibility. England et al.89 synthesised polypyrrole superstructures within the solvent channels of CLLCs in a reproducible and inexpensive approach. The well prepared protein–polymer cross-linked nano-arrays were electrically conductive and exhibited excellent mechanically plasticity. Tabe et al.90 prepared a nano-material by incorporating ruthenium complexes into porous CLLCs. This nano-material showed high catalytic activity in the reaction of acetophenone and its derivatives.
5.1.2 Feasibility study of protein crystal as bio-templates. Protein crystals possess a three-dimensional ordered arrangement of protein molecules. Corresponding to the ordered array of molecules, the resultant interconnected holes can accommodate small molecules. Cvetkovic et al.91 investigated the transport of fluorescein in lysozyme crystals by confocal laser scanning microscopy and found that the diffusion rate was highly related to the crystal structure. Based on these properties, the application of CLPCs in organic synthesis62 and size exclusion chromatography53 has already been developed and improved. Currently, the CLPCs have attracted a great deal of research interest as biological templates for the preparation of novel nano-structured composite materials.78

In 2003, Turner92 first described the utilisation of sturdy CLPCs as the templates for the infusion of metal and polymer loaded nano-composites. Since then, the feasibility of using CLPCs as effective bio-templates for the construction of novel patterned composite materials has attracted pronounced attention.93 Cohen et al.94 dissolved CLPCs in aqueous ethylene glycol, induced polymerisation by the addition of ammonium persulfate, and then retrieved the crystals. The entire process was monitored by step-by-step structural analysis, visualising the elaborate changes in the 3D array of protein molecules throughout the process. This work demonstrated the feasibility of using stabilised protein crystals as the bio-template for the fabrication of nanostructures. Based on previous work, they studied cross-linked lysozyme and concanavalin A crystals as templates for the preparation of silver particles, providing further evidence for the bio-template provided by the protein crystals.95 Additionally, throughout the crystallization process, Wine et al.96 investigated the influence of site specific mutations of lysine residues located in the protein crystal on crystal porosity and their application as bio-templates. Cohen-Hadar et al.97 obtained protein crystals of different porosities (providing various sizes of interconnected voids) by site directed chemical modification of the lysine residues on the protein surface, and they proposed the feasibility of using these crystals as bio-templates to construct organic or inorganic materials. Buch et al.98 proposed and demonstrated the feasibility of disentangling homogeneous populations of protein molecules with identical chemical point mutations and protein dimmers with the same residues cross-linked. The quality of the nano-structured composites obtained using CLPCs as bio-templates as well as their potential applications are strongly related to the preservation of the molecular structure of the protein molecules within the crystal throughout every step involved in the synthetic process.94

5.2 Sensing materials for biosensors

Biosensors are analytical devices that detect, record and transmit information about the changes in a biological signal, and then they convert the biochemical signals into electrical signals (Fig. 5). A biosensor device usually includes the sensitive biological element (such as tissues, microorganisms, organelles, cell receptors, proteins, enzymes, DNA, etc.), the detector element (the transducer that transforms the biological signal to the measurable electrical signal), and the sensor reader that displays the results.
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Fig. 5 Schematic description of a typical biosensor.

Biosensors provide a new class of inexpensive, convenient methods that conduct intricate analyses for rapid and specific methods. The performance of the biosensors relies on the activity and sensitivity of the bioactive substance.20 Proteins and enzymes, which are both bioactive substances, can be combined into a biosensor for specific detecting purpose. The CLPCs exhibit high molecule density and high specific activity; hence, they can produce strong electrical signals even for the smallest quantity of analyte, making them highly attractive for the biosensor component.

Because the urea level in the circulatory fluids is an important indicator of renal disease, Navia et al.99 used urease CLECs in a clinical sensor device for the diagnosis of renal disease. Furthermore, detecting blood glucose is important for the diagnosis of diabetes. Luiz et al.100 fixed lyophilized, cross-linked glucose oxidase crystals and lyophilised glucose oxidase on glassy carbon electrodes and prepared two types of glucose biosensors to detect the glucose levels. Their results indicated that glucose oxidase CLECs presented higher sensitivity and better stability than commercial glucose oxidase. Moreover, the performance of the biosensors could be improved by increasing the enzyme concentration on the electrodes.

CLPCs can also be used in detecting organic pollutants. Roy et al.101 embedded laccase CLEC in a 30% polyvinylpropylidone gel, attached them to electrodes, and thus obtained a biosensor to detect the concentration of phenols. This study found that laccase CLECs possess high activity towards phenols. Their sensor had a short response time to lower molecular weight substrates (2-amino phenol, catechol and pyrogallol), while the reaction time was relatively longer in the higher molecular weight substrates (catechin). In addition, the laccase CLECs maintained high activity towards phenols for over 3 months. Laothanachareon et al.102 constructed an amperometric biosensor for detecting organophosphorus compounds using organophosphate hydrolase CLECs and carbon nano-tubes in a simple and economical approach, that exhibited excellent sensitivity and stability compared with this sensor in its non-crystal form.

5.3 Chromatographic analysis

The porosity of the protein crystals makes them potentially attractive as matrices for material separation.73 However, the protein molecules are bond weakly together in crystals. When used to pack a column, protein crystals will damage easily under hydrostatic pressure and dissolve quickly in the aqueous solution. Hence, the protein crystals cannot be used directly for separation.

After cross-linking, CLPCs obtain a solidified structure, possessing high stability and mechanical strength. Thus, they are able to withstand the mechanical perturbations produced during shearing, mixing, filtering, and pumping. Because they are ideal high porosity molecular sieve materials with unique pore size and porosity, CLPCs are meaningful chromatographic stationary phases for size exclusion chromatography, chiral chromatography,103 affinity chromatography, and hydrophobic interaction chromatography. Substance separation can be achieved by flowing the substances through CLPCs at different speeds. The elution order and the quality of separation are correlated with the contacts between the substances to be separated and the CLPCs. The separation methods using CLPCs as the stationary phase material to separate substances include the following four main types:

5.3.1 Size exclusion chromatography. This method is based on the size of the crystal solvent channel and the analyte of interest. The uniform microporous channels fill 30–65% of the entire crystal volume75 and the diameter of the solvent channel changes from 15 to 100 Å, depending on the nature of protein, its source, and the crystallisation conditions.73 In addition, the anisotropic solute diffusivities and the pore size of the protein crystals are linearly correlated,104 and the solvent channels allow molecules of high molecular weight (up to 100[thin space (1/6-em)]000 Daltons) to enter and pass through the crystals, thus separating substances according to their size.
5.3.2 Chiral chromatography. The L-amino acids create an asymmetric environment that can be exploited in the separation of enantiomers. The L-amino acids in the protein molecular structure can combine with the chiral enantiomers by acting as chiral sites. This combination will produce different hydrogen bonding patterns, electrostatic interactions, hydrophobic interactions, and ion pair interactions, among others, to achieve the purpose of separation.
5.3.3 Affinity chromatography. This method is based on the specific affinity between biomolecules. When the CLPCs are used as the stationary phase, different affinities between the analyte and the stationary phase can be used to separate the desired specific component from the impurities.
5.3.4 Hydrophobic interaction chromatography. To achieve separation, this method utilises different hydrophobic interactions between the analyte and the CLPCs due to different surface hydrophobicities of the analyte.

In summary, these CLPCs can be made both chemically and mechanically stable, and they are capable of separating substances via different properties in size, chemical structure, surface properties and chirality. Therefore, CLPCs are now being increasingly utilised as novel separation materials in chromatographic analysis (Table 1).

Table 1 A brief overview of the CLPCs used in chromatographic analysis
Year Authors Cross-linked protein crystals Methods Separated materials Ref. Note
1998 Vilenchik, et al. Thermolysin–CLECs Size exclusion chromatography Polyethylene glycol 73  
Adsorption chromatography Ibuprofen and phenyllactic acid
Chiral chromatography S/R-phenylglycine; S/R-phenyllactic acid
Human serum albumins–CLPCs Chiral chromatography Two enantiomers of folinic acid
1998 Pastinen, et al. Cross-linked glucose isomerase crystals (CLGI) Affinity chromatography Sorbitol, xylitol and ribitol 105  
2000 Pastinen, et al. CLGI Size exclusion chromatography <1000 g mol−1 polyethylene glycols 106  
Hydrophobic interaction chromatography n-alcohols C1 to C8
Chiral chromatography D/L-arabitol
2001 Leisola, et al. Cross-linked xylanase Simultaneous catalysis and separation From xylotetraose/xylotriose to xylobiose/xylose 107  
2002 Jokela et al. Cross-linked xylose isomerase crystals (CLXIC) Chiral chromatography pentose and hexose sugars 16  
2003 Vuolanto, et al. Cross-linked antibody Fab fragment crystals Chiral chromatography A drug enantiomers from racemic mixture 108  
2004 Jokela, et al. CLXIC Affinity chromatography Uridine, cytidine, adenosine, guanosine and thymidine 109  
2009 Hu, et al. Thermolysin crystal Chiral chromatography Racemic phenylglycines 110 Molecular simulations

5.4 Oral delivery

The protein pharmaceutical market is quickly growing, and the oral delivery of drugs has garnered attention because of its simplicity, convenience and high bioavailability. Combined with permeability and stability, the solubility of a drug is also a determining factor for its oral delivery. Protein drugs have been made into different crystalline states, and hydrogels are common protein drug carriers to slow down the release rate of the protein.111

Typically, protein crystals dissolve gradually when transferred from the crystallisation mother liquor to fresh buffer. Such behaviour limits the sustained release capacity of these drugs. Cross-linking the crystal surface or cocrystallization of the protein with ligand are useful approaches to slow down the lattice dissociation rate of the protein molecules from the crystalline state, and to reduce the solubility of the protein crystals in the medium. During the chemical cross-linking of protein crystals by a bifunctional reagent, controlling the surface cross-link density to adjust the dissolution rate of the protein molecules can be used to reach an ideal drug dissolution rate.

When crystalline antigenic protein passes through the gastrointestinal mucosa, it will cause the organism to produce a more effective immune response and immune tolerance than its dissolved form. Burroughs et al.112 reported the immunogenicity of protein crystals and Clair et al.113 concluded that the immunogenicity of cross-linked human serum albumin crystals was better than that of soluble proteins. They showed that, compared with the soluble protein, highly cross-linked and lightly cross-linked human serum albumin crystals induced a 6- to 10-fold and 30-fold increase in antibody titer, respectively. Apart from the immunogenicity, other features of CLPCs are also beneficial for drug delivery. Margolin et al.114 prepared cross-linked porcine pancreatic lipase crystals. Their stability and activity were increased 100 times compared with the ordinary enzyme; hence, their resistance to gastric acid and digestive enzymes in the gastrointestinal tract was greatly improved, which made them a perfect choice for the treatment for digestive disorders.

Anjani et al.115 incorporated soluble enzymes into polymer matrices (alginate, carrageenan, agar, etc.), and found that the enzymes would leak out of the matrix if the pore size of the polymer matrices was larger than the enzyme molecules. Taking advantage of the larger size of cross-linked enzyme crystals compared to normal hydrogels, Simi et al.116 encapsulated a protease CLEC in a natural hydrogel to control, for the first time, the release rate of proteins for oral delivery. A slow release velocity ensures long-term efficacy of the drugs, and the gel microspheres have sensitivity to pH, which could contribute to protecting the protein from the damage of gastric juice. The remarkable features of CLPCs, such as enhanced immunogenicity, stability, activity, and size make them perfect candidates for vaccine formulations.

5.5 Quality of CLPCs

Currently, X-ray diffraction is the primary technique to obtain the three dimensional structure of protein molecules. However, the crystals to be diffracted are usually fragile and hard to handle. Certain crystals cannot be directly placed in antifreeze; therefore, it is difficult for these crystals to be used for diffraction analysis. To tackle this problem, several post-crystallisation treatments, such as crystal annealing,117,118 crystal dehydration119 and CLPC technology, have been proposed. Among these treatments, CLPC technology promotes the formation of strong covalent bonds between the protein molecules and produces crystals with high quality. The diffraction quality of the CLPCs can also be improved,120 particularly for fragile protein crystals.

There have been several successful reports about improving the quality of protein crystals by limited glutaraldehyde treatment. Lusty34 found that the diffraction patterns of an N-cadherin fragment crystal and a reverse transcriptase-DNA complex crystal remained the same after chemical cross-linking, but for a gp120 ternary complex crystal, significantly better diffraction resolution (2.2 Å) than that without cross-linking (2.7 Å) was observed. In addition, the mosaicity of the crystal was strikingly decreased after cross-linking by glutaraldehyde. Remenyi et al.121 showed that the resolution of protein–DNA complex crystals increased by 1.3 Å, while the mosaicity reduced by 1.5° after cross-linking. Duan et al.122 showed that the diffraction data of native crystals and SeMet crystals improved notably from 5 Å to 2.3 Å and 7 Å to 3.9 Å respectively, when diffracted at a home X-ray source and a synchrotron before and after cross-linking. In both cases, the mosaicity decreased dramatically. Chemical cross-linking will result in slight changes in the three-dimensional structure of particles, thus the lattice constants can be somewhat variable. Limura et al.36 found that the a- and b-axis increased by approximately 0.04 Å in lysozyme crystals after chemical cross-linking, while the c-axis lattice parameters reduced by 0.16 Å. Fortunately, though the lattice constants of the CLPCs can be a little variable due to the chemical cross-linking, the diffraction pattern of the CLPCs is very similar to that of the native crystals, and there are no detectable20 or only minor differences22 in protein structure after cross-linking.

Apart from the successful examples, there are also reports showing no obvious effects by cross-linking. Quiocho et al.24 found that the X-ray diffraction pattern of cross-linked carboxypeptidase-A crystals was similar to the native crystals. Kaufmann et al.123 found that the treatment of West Nile virus crystals using glutaraldehyde showed no recognisable effect on crystal morphology or the diffraction pattern. Andersen et al.124 found that the resolution of cross-linked hPDE10a crystals decreased by 0.5 Å to 2.8 Å when compared with native crystals (2.3 Å).

6 Concluding remarks

6.1 Limitations

With the continuous development of protein crystallisation methodology and cross-linking technology, an increasing number of proteins have been made into CLPCs and their applications have been gradually broadened. However, there are still some limitations for development in this field, which require further research to acquire a solution.

(1) The preparation of protein crystals requires an abundance of high purity protein. It is relatively easy to meet the harsh crystallisation conditions in a laboratory, however it is not that easy to do in industry. Currently, the pure proteins commercially available for CLPCs are limited. Furthermore, there is a great loss of proteins in the purification, crystallisation and cross-linking processes. Hence, efficient methods for protein expression, purification and crystallisation methods are necessary.

(2) The cross-linking status is usually correlated with the location in the crystals. The outer layer of CLPCs is frequently excessively cross-linked, while in the internal part, the cross-linking is still partial. This cross-linking in homogeneity can cause the crystals to crack in buffer. New methods to solve this problem should be developed.

(3) The mechanism of cross-linking of CLPCs is still presently unclear. It is necessary to explore the mechanism in more detail so that it can be applied to study the applications of CLPCs.

6.2 Perspective

Despite the limitations, the advantages of CLPCs are clear and superior to untreated crystals. With the development of protein crystallogenesis and cross-linking techniques, CLPCs show better performance at less cost, which makes their applications more attractive in materials processing, biosensors, chromatographic analysis, oral drug delivery, and the improvement of crystals quality. Furthermore, new applications of CLPCs may be elucidated in the near future following extensive investigation.


This work was supported by National Basic Research Program of China (973 Program, Grant no. 2011CB710905), National Natural Science Foundation of China (Grant nos 31170816, 11202167, and 51201137), the Fundamental Research Funds for the Central Universities (Grant nos 3102014JKY15006 and 3102014KYJD019).

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