Energetic Changes Caused by Antigenic Module Insertion in a Virus-Like Particle Revealed by Experiment and Molecular Dynamics Simulations

The success of recombinant virus-like particles (VLPs) for human papillomavirus and hepatitis B demonstrates the potential of VLPs as safe and efficacious vaccines. With new modular designs emerging, the effects of antigen module insertion on the self-assembly and structural integrity of VLPs should be clarified so as to better enabling improved design. Previous work has revealed insights into the molecular energetics of a VLP subunit, capsomere, comparing energetics within various solution conditions known to drive or inhibit self-assembly. In the present study, molecular dynamics (MD) simulations coupled with the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method were performed to examine the molecular interactions and energetics in a modular capsomere of a murine polyomavirus (MPV) VLP designed to protect against influenza. Insertion of an influenza antigenic module is found to lower the binding energy within the capsomere, and a more active state is observed in Assembly Buffer as compared with that in Stabilization Buffer, which has been experimentally validated through measurements using differential scanning calorimetry. Further in-depth analysis based on free-energy decomposition indicates that destabilized binding can be attributed to electrostatic interaction induced by the chosen antigen module. These results provide molecular insights into the conformational stability of capsomeres and their abilities to be exploited for antigen presentation, and are expected to be beneficial for the biomolecular engineering of VLP vaccines.     

Engineering Tobacco Mosaic Virus and Its Virus-Like-Particles for Synthesis of Biotemplated Nanomaterials

Biomolecules are increasingly attractive templates for the synthesis of functional nanomaterials. Chief among them is the plant tobacco mosaic virus (TMV) due to its high aspect ratio, narrow size distribution, diverse biochemical functionalities presented on the surface, and compatibility with a number of chemical conjugations. These properties are also easily manipulated by genetic modification to enable the synthesis of a range of metallic and non-metallic nanomaterials for diverse applications. This article reviews the characteristics of TMV and related viruses, and their virus-like particle (VLP) derivatives, and how these may be manipulated to extend their use and function. A focus of recent efforts has been on greater understanding and control of the self-assembly processes that drive biotemplate formation. How these features have been exploited in engineering applications such as, sensing, catalysis, and energy storage are briefly outlined. While control of VLP surface features is well-established, fewer tools exist to control VLP self-assembly, which limits efforts to control template uniformity and synthesis of certain templated nanomaterials. However, emerging advances in synthetic biology, machine learning, and other fields promise to accelerate efforts to control template uniformity and nanomaterial synthesis enabling more widescale industrial use of VLP-based biotemplates.     

Challenges for the production of virus-like particles in insect cells: The case of rotavirus-like particles

Virus-like particles (VLP) are formed when viral structural proteins are produced in an heterologous expression system. Such proteins assemble into structures that are morphologically similar to native viruses but lack the viral genome. VLP are complex structures with a wide variety of applications, ranging from basic research and vaccines to potential new uses in nanotechnology. Production of VLP is a challenging task, as both the synthesis and assembly of one or more recombinant proteins are required. This is the case for VLP of rotavirus (RLP), which is an RNA virus with a capsid formed by 1860 monomers of four different proteins. In addition, the production of most VLP requires the simultaneous expression and assembly of several recombinant proteins, which – for the case of RLP – needs to occur in a single host cell. The insect cell baculovirus expression vector system (IC-BEVS) has been shown to be a powerful and convenient system for rapidly and easily producing VLP, due to several convenient features, including its versatility and the short time needed for construction of recombinant baculovirus. In this review, the specific case of rotavirus-like particle (RLP) production by the IC-BEVS is discussed, with emphasis on bioprocess engineering issues that exist and their solutions. Many culture strategies discussed here can be useful for the production of other VLP.     

Layer-by-layer self-assembly of supramolecular and biomolecular films

In this paper, we give a short account on recent studies of layer-by-layer self-assembly of supramolecular and biomolecular films. Such films are built up from layers of macro-ions with opposing charge. A simple film can be obtained by alternating the adsorption of two components: a flexible, synthetic polycation chains and a supramolecular or biomolecular moiety. We focus on three examples, in which the second component consists either of a supramolecular metal-organic complex (MOC), a nucleic acid, or a biological membrane patch (purple membrane). While the flexible polvcation chains (as well as eventual annealing layers) ensure a uniform build-up of the chain, the second macromolecular component may be used to functionalize the films. The combination of layer-by-layer self-assembly and biotechnologically relevant macromolecules may lead to new devices or biomaterial applications. To this end, precise studies of the deposition process and the film structure are needed. Here, we focus on interface sensitive scattering techniques for the structural analysis.     

RNA Control of HIV-1 Particle Size Polydispersity

HIV-1, an enveloped RNA virus, produces viral particles that are known to be much more heterogeneous in size than is typical of non-enveloped viruses. We present here a novel strategy to study HIV-1 Viral Like Particles (VLP) assembly by measuring the size distribution of these purified VLPs and subsequent viral cores thanks to Atomic Force Microscopy imaging and statistical analysis. This strategy allowed us to identify whether the presence of viral RNA acts as a modulator for VLPs and cores size heterogeneity in a large population of particles. These results are analyzed in the light of a recently proposed statistical physics model for the self-assembly process. In particular, our results reveal that the modulation of size distribution by the presence of viral RNA is qualitatively reproduced, suggesting therefore an entropic origin for the modulation of RNA uptake by the nascent VLP.     

Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles.

The L1 genes of two human papillomavirus type 16 (HPV16) isolates derived from condylomata acuminata were used to express the L1 major capsid protein in insect cells via recombinant baculoviruses. Both L1 major capsid proteins self-assembled into virus-like particles (VLP) with high efficiency and could be purified in preparative amounts on density gradients. The yield of VLP was 3 orders of magnitude higher than what has been obtained previously, using L1 derived from the prototype HPV16. DNA sequence comparison identified a single nonconserved amino acid change to be responsible for the inefficient self-assembly of the prototype L1. VLP were also obtained by expressing L1 of HPV6, HPV11, and cottontail rabbit papillomavirus, indicating that L1 from a variety of papillomaviruses has the intrinsic capacity to self-assemble into VLP. Coexpression of HPV16 L1 plus L2 by using a baculovirus double-expression vector also resulted in efficient self-assembly of VLP, and the average particle yield increased about fourfold in comparison to when L1 only was expressed. Coimmunoprecipitation of L1 and L2 and cosedimentation of the two proteins in a sucrose gradient demonstrated that L2 was incorporated into the particles. The ability to generate preparative amounts of HPV16 L1 and L1-L2 VLP may have implications for the development of a serological assay to detect anti-HPV16 virion immune responses to conformational epitopes and for immunoprophylaxis against HPV16 infection.     

Recent progress in biomolecular engineering

During the next decade or so, there will be significant and impressive advances in biomolecular engineering, especially in our understanding of the biological roles of various biomolecules inside the cell. The advances in high throughput screening technology for discovery of target molecules and the accumulation of functional genomics and proteomics data at accelerating rates will enable us to design and discover novel biomolecules and proteins on a rational basis in diverse areas of pharmaceutical, agricultural, industrial, and environmental applications. As an applied molecular evolution technology, DNA shuffling will play a key role in biomolecular engineering. In contrast to the point mutation techniques, DNA shuffling exchanges large functional domains of sequences to search for the best candidate molecule, thus mimicking and accelerating the process of sexual recombination in the evolution of life. The phage-display system of combinatorial peptide libraries will be extensively exploited to design and create many novel proteins, as a result of the relative ease of screening and identifying desirable proteins. Even though this system has so far been employed mainly in screening the combinatorial antibody libraries, its application will be extended further into the science of protein-receptor or protein-ligand interactions. The bioinformatics for genome and proteome analyses will contribute substantially toward ever more accelerated advances in the pharmaceutical industry. Biomolecular engineering will no doubt become one of the most important scientific disciplines, because it will enable systematic and comprehensive analyses of gene expression patterns in both normal and diseased cells, as well as the discovery of many new high-value molecules. When the functional genomics database, EST and SAGE techniques, microarray technique, and proteome analysis by 2-dimensional gel electrophoresis or capillary electrophoresis in combination with mass spectrometer are all put to good use, biomolecular engineering research will yield new drug discoveries, improved therapies, and significantly improved or new bioprocess technology. With the advances in biomolecular engineering, the rate of finding new high-value peptides or proteins, including antibodies, vaccines, enzymes, and therapeutic peptides, will continue to accelerate. The targets for the rational design of biomolecules will be broad, diverse, and complex, but many application goals can be achieved through the expansion of knowledge based on biomolecules and their roles and functions in cells and tissues. Some engineered biomolecules, including humanized Mab's, have already entered the clinical trials for therapeutic uses. Early results of the trials and their efficacy are positive and encouraging. Among them, Herceptin, a humanized Mab for breast cancer treatment, became the first drug designed by a biomolecular engineering approach and was approved by the FDA. Soon, new therapeutic drugs and high-value biomolecules will be designed and produced by biomolecular engineering for the treatment or prevention of not-so-easily cured diseases such as cancers, genetic diseases, age-related diseases, and other metabolic diseases. Many more industrial enzymes, which will be engineered to confer desirable properties for the process improvement and manufacturing of high-value biomolecular products at a lower production cost, are also anticipated. New metabolites, including novel antibiotics that are active against resistant strains, will also be produced soon by recombinant organisms having de novo engineered biosynthetic pathway enzyme systems. The biomolecular engineering era is here, and many of benefits will be derived from this field of scientific research for years to come if we are willing to put it to good use.     

Hantavirus Gn and Gc Glycoproteins Self-Assemble into Virus-Like Particles

How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera.     

Induction of protective immune response to intranasal administration of influenza virus-like particles in a mouse model

Human influenza A viruses (IAVs) cause global pandemics and epidemics, which remains a nonignorable serious concern for public health worldwide. To combat the surge of viral outbreaks, new treatments are urgently needed. Here, we design a new vaccine based on virus-like particles (VLPs) and show how intranasal administration of this vaccine triggers protective immunity, which can be exploited for the development of new therapies. H1N1 VLPs were produced in baculovirus vectors and were injected into BALB/c mice by the intramuscular (IM) or intranasal (IN) route. We found that there were significantly higher inflammatory cell and lymphocyte concentrations in bronchoalveolar lavage samples and the lungs of IN immunized mice; however, the IM group had little signs of inflammatory responses. On the basis of our results, immunization with H1N1 influenza VLP elicited a strong T cell immunity in BALB/c mice. Despite T cell immunity amplification after both IN and IM vaccination methods in mice, IN-induced T cell responses were significantly more intense than IM-induced responses, and this was likely related to an increased number of both CD11b^high and CD103⁺ dendritic cells in mice lungs after IN administration of VLP. Furthermore, evaluation of interleukin-4 and interferon gamma cytokines along with several chemokine receptors showed that VLP vaccination via IN and IM routes leads to a greater CD4⁺ Th1 and Th2 response, respectively. Our findings indicated that VLPs represent a potential strategy for the development of an effective influenza vaccine; however, employing relevant routes for vaccination can be another important part of the universal influenza vaccine puzzle.     

Quantitative characterization of virus-like particles by asymmetrical flow field flow fractionation, electrospray differential mobility analysis, and transmission electron microscopy

Here we characterize virus-like particles (VLPs) by three very distinct, orthogonal, and quantitative techniques: electrospray differential mobility analysis (ES-DMA), asymmetric flow field-flow fractionation with multi-angle light scattering detection (AFFFF-MALS) and transmission electron microscopy (TEM). VLPs are biomolecular particles assembled from viral proteins with applications ranging from synthetic vaccines to vectors for delivery of gene and drug therapies. VLPs may have polydispersed, multimodal size distributions, where the size distribution can be altered by subtle changes in the production process. These three techniques detect subtle size differences in VLPs derived from the non-enveloped murine polyomavirus (MPV) following: (i) functionalization of the surface of VLPs with an influenza viral peptide fragment; (ii) packaging of foreign protein internally within the VLPs; and (iii) packaging of genomic DNA internally within the VLPs. These results demonstrate that ES-DMA and AFFFF-MALS are able to quantitatively determine VLP size distributions with greater rapidity and statistical significance than TEM, providing useful technologies for product development and process analytics.     

Intranasal delivery of recombinant parvovirus-like particles elicits cytotoxic T-cell and neutralizing antibody responses

We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4(+) and CD8(+) T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8(+) T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in the absence of adjuvant, developed high levels of PPV-specific immunoglobulin G (IgG) and/or IgA in their serum, as well as in mucosal sites such as the bronchoalveolar and intestinal fluids. Antibodies in sera from mice immunized parenterally or intranasally with PPV:VLP were strongly neutralizing in vitro. Intranasal immunization with PPV:VLP carrying the LCMV CD8(+) T-cell epitope also elicited a strong peptide-specific cytotoxic-T-cell (CTL) response. In contrast, mice orally immunized with recombinant PPV:VLP did not develop any antibody or CTL responses. We also showed that mice primed with PPV:VLP are still able to develop strong CTL responses after subsequent immunization with chimeric PPV:VLP carrying a foreign CD8(+) T-cell epitope. These results highlight the attractive potential of PPV:VLP as a safe, nonreplicating antigen carrier to stimulate systemic and mucosal immunity after nasal administration.     

鼠多瘤病毒样颗粒及其在疫苗开发等方面的研究进展

鼠多瘤病毒(murine polyomavirus,MPV)的病毒样颗粒(virus-like particle,VLP)是通过MPV的衣壳结构蛋白VP1自组装而成的球形纳米壳状结构. MPV VLP具有独特的纳米结构,在一定条件下能够进行体内或体外自组装,具有丰富的可修饰位点.因此,容易通过结构的修饰改造实现MPV VLP在诸多领域的应用.本文从MPV VLP的结构特点着眼,回顾MPV VLP的发现历程,介绍MPV VLP的制备表达系统和组装机理,综述MPV VLP的修饰.重点介绍化学修饰和基因工程修饰方法,并通过实例阐述MPV VLP在疫苗开发、药物及其他分子载体等领域的应用及其研究进展.基于对已有研究进展的分析,指出大规模生产、组装机理解析及其应用研究的关键环节,服务于MPV VLP的应用开发.     

Role for amino acids 212KLR214 of Ebola virus VP40 in assembly and budding

Ebola virus VP40 is able to produce virus-like particles (VLPs) in the absence of other viral proteins. At least three domains within VP40 are thought to be required for efficient VLP release: the late domain (L-domain), membrane association domain (M-domain), and self-interaction domain (I-domain). While the L-domain of Ebola VP40 has been well characterized, the exact mechanism by which VP40 mediates budding through the M- and I-domains remains unclear. To identify additional domains important for VP40 assembly/budding, amino acids (212)KLR(214) were targeted for mutagenesis based on the published crystal structure of VP40. These residues are part of a loop connecting two beta sheets in the C-terminal region and thus are potentially important for overall structure and/or oligomerization of VP40. A series of alanine substitutions were generated in the KLR region of VP40, and these mutants were examined for VLP budding, intracellular localization, and oligomerization. Our results indicated that (i) (212)KLR(214) residues of VP40 are important for efficient release of VP40 VLPs, with Leu213 being the most critical; (ii) VP40 KLR mutants displayed altered patterns of cellular localization compared to that of wild-type VP40 (VP40-WT); and (iii) self-assembly of VP40 KLR mutants into oligomers was altered compared to that of VP40-WT. These results suggest that (12)KLR(214) residues of VP40 are important for proper assembly/oligomerization of VP40 which subsequently leads to efficient budding of VLPs.     

Peptoids at the 7th Summit: toward macromolecular systems engineering

Methods for facile synthesis of extraordinarily diverse peptide-like oligomers have placed peptoids at the center of a broad and vibrant area of foldamer science and technology. The 7th Peptoid Summit offered a perspective on the current state of peptoid science and technology and on prospects for engineering supramolecular assemblies that rival the complexity of biomolecular systems. Methods for engineering biomolecular systems based on DNA and protein are advancing rapidly, building a technology platform for engineering increasingly large and complex self-assembled nanosystems. A comparative review of the physical basis for DNA, protein, and peptoid engineering indicates that the characteristics of peptoids suit them for a strong role in developing self-assembled nanosystems. Physical parallels between peptoids and proteins indicate that peptoid engineering, like protein engineering, will require specialized software to support design. Access to novel side-chain functionality will enable peptoid designers to exploit novel binding interactions, including many that have been discovered and exploited in crystal engineering, a field that has extensively explored the self-assembly of small organic molecules to form well-ordered structures. Developments in DNA, protein, and inorganic nanotechnologies are converging to provide a technology platform for the design and fabrication of complex, functional, atomically precise nanosystems. Peptoid-based foldamer technologies, can contribute to this convergence, expanding the scope of the emerging field of atomically precise macromolecular nanosystems.     

Different sequences show similar quaternary interaction stabilities in prohead viral RNA self-assembly

Prohead RNA (pRNA) is an essential component of the self-assembling φ29 bacteriophage DNA packaging motor. Different related species of bacteriophage share only 12% similarity in pRNA sequences. The secondary structure for pRNA is conserved, however. In this study, we present evidence for self-assembly in different pRNA sequences and new measurements of the energetics for the quaternary interactions in pRNA dimers and trimers. The energetics for self-assembly in different pRNA sequences are similar despite very different sequences in the loop-loop interactions. The architecture surrounding the interlocking loops contributes to the stability of the pRNA quaternary interactions, and sequence variation outside the interlocking loops may counterbalance the changes in the loop sequences. Thus, the evolutionary divergence of pRNA sequences maintains not only conservation of function and secondary structure but also stabilities of quaternary interactions. The self-assembly of pRNA can be fine-tuned with variations in magnesium chloride, sodium chloride, temperature, and concentration. The ability to control pRNA self-assembly holds promise for the development of nanoparticle therapeutic applications for this biological molecule. The pRNA system is well suited for future studies to further understand the energetics of RNA tertiary and quaternary interactions, which can provide insight into larger biological assemblies such as viruses and biomolecular motors.     

Influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmid-derived virus-like particles

For influenza virus, we developed an efficient, noncytotoxic, plasmid-based virus-like particle (VLP) system to reflect authentic virus particles. This system was characterized biochemically by analysis of VLP protein composition, morphologically by electron microscopy, and functionally with a VLP infectivity assay. The VLP system was used to address the identity of the minimal set of viral proteins required for budding. Combinations of viral proteins were expressed in cells, and the polypeptide composition of the particles released into the culture media was analyzed. Contrary to previous findings in which matrix (M1) protein was considered to be the driving force of budding because M1 was found to be released copiously into the culture medium when M1 was expressed by using the vaccinia virus T7 RNA polymerase-driven overexpression system, in our noncytotoxic VLP system M1 was not released efficiently into the culture medium. Additionally, hemagglutinin (HA), when treated with exogenous neuraminidase (NA) or coexpressed with viral NA, could be released from cells independently of M1. Incorporation of M1 into VLPs required HA expression, although when M1 was omitted from VLPs, particles with morphologies similar to those of wild-type VLPs or viruses were observed. Furthermore, when HA and NA cytoplasmic tail mutants were included in the VLPs, M1 failed to be efficiently incorporated into VLPs, consistent with a model in which the glycoproteins control virus budding by sorting to lipid raft microdomains and recruiting the internal viral core components. VLP formation also occurred independently of the function of Vps4 in the multivesicular body pathway, as dominant-negative Vps4 proteins failed to inhibit influenza VLP budding.     

基于表面等离子共振的新型生物传感技术及其在生命科学中的应用

生物分子的活性功能是通过分子之间的相互作用来体现的,了解这种相互作用的过程对于生命科学领域的研究及揭示生命发生发展的基本机制具有重要的意义。基于表面等离子共振(surface plasmon resonance,SPR)的新型生物传感技术——BIAcore(biomolecular interaction analysis)是研究生物分子相互作用的理想工具。它可以实时跟踪检测生物分子间结合、解离的整个过程,已被广泛应用于蛋白质组学、信号转导、新药开发、遗传学分析和食品检测等领域,并且显示出广阔的应用前景。     

Time-Resolved study of network self-organization from a biopolymeric solution

Time-resolved studies of network self-organization from homogeneous solutions of the representative biostructural polymer agarose are presented. Solutions are temperature quenched and observed by several techniques. Consistent with previous suggestions by the authors, experiments at concentrations up to about 1.75% w/v provide direct kinetic evidence for the occurrence of at least two distinct processes, leading, in sequence, to self-assembly. These are as follows: (a) a liquid–liquid phase separation of the solution occurring via spinodal demixing and resulting in two sets of regions that have, respectively, higher and lower than average concentrations of random-coiled polymers; and (b) the subsequent 2 coils → double helix transition and accompanying cross-linking and gelation (due to branching of double helices), occurring in the high-concentration regions. The size of the high-concentration regions depends upon agarose concentration and quenching temperature, and is in the range from a fraction of micrometers to a few micrometers, in agreement with earlier experiments. Bundling of the double-helical segments is known to follow self-assembly and can be considered as a third step (gel curing). This follows from the thermo-dynamic instability of the helical segments in the solvent, behaving as a system of rod-like particles connected by more or less flexible joints. The two processes leading in succession to self-assembly are discussed in terms of a phase diagram consistent with available data. Their time scales differ remarkably. At the end of the first process, all polymers remain random coiled and freely drifting. Much later coil–helix transition is observed, always in coincidence with polymer cross-linking and gelation. The enhancement of concentration of random-coiled polymers in specific regions of the sol caused by spinodal demixing is thus a prerequisite for self-assembly of these biostructural gels in the concentration interval studied. Conceptually, concentration enhancements of this type can provide a new pathway for promotion of functional biomolecular interactions even at very low average concentrations. The mechanism will work identically if the region of instability is reached by varying the polymer concentration (e.g., by biosynthesis), rather than by temperature quenching.     

Synthetic biology for bioengineering virus-like particle vaccines

Vaccination is the most effective method of disease prevention and control. Many viruses and bacteria that once caused catastrophic pandemics (e.g., smallpox, poliomyelitis, measles, and diphtheria) are either eradicated or effectively controlled through routine vaccination programs. Nonetheless, vaccine manufacturing remains incredibly challenging. Viruses exhibiting high antigenic diversity and high mutation rates cannot be fairly contested using traditional vaccine production methods and complexities surrounding the manufacturing processes, which impose significant limitations. Virus-like particles (VLPs) are recombinantly produced viral structures that exhibit immunoprotective traits of native viruses but are noninfectious. Several VLPs that compositionally match a given natural virus have been developed and licensed as vaccines. Expansively, a plethora of studies now confirms that VLPs can be designed to safely present heterologous antigens from a variety of pathogens unrelated to the chosen carrier VLPs. Owing to this design versatility, VLPs offer technological opportunities to modernize vaccine supply and disease response through rational bioengineering. These opportunities are greatly enhanced with the application of synthetic biology, the redesign and construction of novel biological entities. This review outlines how synthetic biology is currently applied to engineer VLP functions and manufacturing process. Current and developing technologies for the identification of novel target-specific antigens and their usefulness for rational engineering of VLP functions (e.g., presentation of structurally diverse antigens, enhanced antigen immunogenicity, and improved vaccine stability) are described. When applied to manufacturing processes, synthetic biology approaches can also overcome specific challenges in VLP vaccine production. Finally, we address several challenges and benefits associated with the translation of VLP vaccine development into the industry.     

Insertional protein engineering for analytical molecular sensing

The quantitative detection of low analyte concentrations in complex samples is becoming an urgent need in biomedical, food and environmental fields. Biosensors, being hybrid devices composed by a biological receptor and a signal transducer, represent valuable alternatives to non biological analytical instruments because of the high specificity of the biomolecular recognition. The vast range of existing protein ligands enable those macromolecules to be used as efficient receptors to cover a diversity of applications. In addition, appropriate protein engineering approaches enable further improvement of the receptor functioning such as enhancing affinity or specificity in the ligand binding. Recently, several protein-only sensors are being developed, in which either both the receptor and signal transducer are parts of the same protein, or that use the whole cell where the protein is produced as transducer. In both cases, as no further chemical coupling is required, the production process is very convenient. However, protein platforms, being rather rigid, restrict the proper signal transduction that necessarily occurs through ligand-induced conformational changes. In this context, insertional protein engineering offers the possibility to develop new devices, efficiently responding to ligand interaction by dramatic conformational changes, in which the specificity and magnitude of the sensing response can be adjusted up to a convenient level for specific analyte species. In this report we will discuss the major engineering approaches taken for the designing of such instruments as well as the relevant examples of resulting protein-only biosensors.