Cloning strategy for producing brush-forming protein-based polymers

Brush-forming polymers are being used in a variety of applications, and by using recombinant DNA technology, there exists the potential to produce protein-based polymers that incorporate unique structures and functions in these brush layers. Despite this potential, production of protein-based brush-forming polymers is not routinely performed. For the design and production of new protein-based polymers with optimal brush-forming properties, it would be desirable to have a cloning strategy that allows an iterative approach wherein the protein based-polymer product can be produced and evaluated, and then if necessary, it can be sequentially modified in a controlled manner to obtain optimal surface density and brush extension. In this work, we report on the development of a cloning strategy intended for the production of protein-based brush-forming polymers. This strategy is based on the assembly of modules of DNA that encode for blocks of protein-based polymers into a commercially available expression vector; there is no need for custom-modified vectors and no need for intermediate cloning vectors. Additionally, because the design of new protein-based biopolymers can be an iterative process, our method enables sequential modification of a protein-based polymer product. With at least 21 bacterial expression vectors and 11 yeast expression vectors compatible with this strategy, there are a number of options available for production of protein-based polymers. It is our intent that this strategy will aid in advancing the production of protein-based brush-forming polymers.     

Engineering of ribozyme-based riboswitches for mammalian cells

Artificial RNA riboswitches--apart from protein-based gene regulation systems, which have been known about for a long time--have become increasingly important in biotechnology and synthetic biology. Aptamer-controlled hammerhead ribozymes (so-called aptazymes) have been shown to be a versatile platform for the engineering of novel gene regulators. Since aptazymes are cis-acting elements that are located in the untranslated regions of a gene of interest, their application does not need any further protein co-factor. This presents the opportunity to simplify complex gene networks while simultaneously expanding the repertoire of available parts. Nevertheless, the generation of novel aptazymes requires a functional aptamer-ribozyme connection, which can be difficult to engineer. This article describes a novel approach for using fluorescence activated cell sorting (FACS) in order to identify functional aptazymes in bacteria and their subsequent transfer into mammalian cells.     

Production of protein-based polymers in Pichia pastoris

Materials science and genetic engineering have joined forces over the last three decades in the development of so-called protein-based polymers. These are proteins, typically with repetitive amino acid sequences, that have such physical properties that they can be used as functional materials. Well-known natural examples are collagen, silk, and elastin, but also artificial sequences have been devised. These proteins can be produced in a suitable host via recombinant DNA technology, and it is this inherent control over monomer sequence and molecular size that renders this class of polymers of particular interest to the fields of nanomaterials and biomedical research. Traditionally, Escherichia coli has been the main workhorse for the production of these polymers, but the methylotrophic yeast Pichia pastoris is finding increased use in view of the often high yields and potential bioprocessing benefits. We here provide an overview of protein-based polymers produced in P. pastoris. We summarize their physicochemical properties, briefly note possible applications, and detail their biosynthesis. Some challenges that may be faced when using P. pastoris for polymer production are identified: (i) low yields and poor process control in shake flask cultures; i.e., the need for bioreactors, (ii) proteolytic degradation, and (iii) self-assembly in vivo. Strategies to overcome these challenges are discussed, which we anticipate will be of interest also to readers involved in protein expression in P. pastoris in general.     

Advances in Understanding Stimulus-Responsive Phase Behavior of Intrinsically Disordered Protein Polymers

Proteins and synthetic polymers can undergo phase transitions in response to changes to intensive solution parameters such as temperature, proton chemical potentials (pH), and hydrostatic pressure. For proteins and protein-based polymers, the information required for stimulus-responsive phase transitions is encoded in their amino acid sequence. Here, we review some of the key physical principles that govern the phase transitions of archetypal intrinsically disordered protein polymers (IDPPs). These are disordered proteins with repetitive amino acid sequences. Advances in recombinant technologies have enabled the design and synthesis of protein sequences of a variety of sequence complexities and lengths. We summarize insights that have been gleaned from the design and characterization of IDPPs that undergo thermo-responsive phase transitions and build on these insights to present a general framework for IDPPs with pH and pressure responsive phase behavior. In doing so, we connect the stimulus-responsive phase behavior of IDPPs with repetitive sequences to the coil-to-globule transitions that these sequences undergo at the single-chain level in response to changes in stimuli. The proposed framework and ongoing studies of stimulus-responsive phase behavior of designed IDPPs have direct implications in bioengineering, where designing sequences with bespoke material properties broadens the spectrum of applications, and in biology and medicine for understanding the sequence-specific driving forces for the formation of protein-based membraneless organelles as well as biological matrices that act as scaffolds for cells and mediators of cell-to-cell communication.     

Sugar-derived glasses support thermal and photo-initiated electron transfer processes over macroscopic distances

Trehalose-derived glasses are shown to support long range electron transfer reactions between spatially well separated donors and protein acceptors. The results indicate that these matrices can be used not only to greatly stabilize protein structures but also to facilitate both thermal and photo-initiated hemeprotein reduction over large macroscopic distances. To date the promise of exciting new protein-based technologies that can harness the exceptional tunability of protein functionality has been significantly thwarted by both intrinsic instability and stringent solvent/environment requirements for the expression of functional properties. The presented results raise the prospect of overcoming these limitations with respect to incorporating redox active proteins into solid state devices such as tunable batteries, switches, and solar cells. The findings also have implications for formulations intended to enhance long term storage of biomaterials, new protein-based synthetic strategies, and biophysical studies of functional intermediates trapped under nonequilibrium conditions. In addition, the study shows that certain sugars such as glucose or tagatose, when added to redox-inactive glassy matrices, can be used as a source of thermal electrons that can be harvested by suitable redox active proteins, raising the prospect of using common sugars as an electron source in solid state thermal fuel cells.     

Conformational behavior of chemically reactive alanine-rich repetitive protein polymers

The synthesis of protein-based polymers with controlled conformational properties and functional group placement offers many opportunities for the design of advanced materials. In this work, protein engineering methods have been used to produce repetitive alanine-rich protein polymers with the sequence [(AAAQ)(5)(AAAE)(AAAQ)(5)](x) (x = 2 and 6); these macromolecules may mimic architectural features of certain alanine-rich helical sequences found in natural proteins. Various proteins from this family can be readily expressed and purified from Escherichia coli. Circular dichroic spectroscopy (CD) characterization demonstrates that the purified proteins are highly helical under a variety of conditions. Thermal analysis of [(AAAQ)(5)(AAAE)(AAAQ)(5)](2) via differential scanning calorimetry (DSC) and CD indicates that the protein undergoes a reversible helix-coil transition at approximately 45 degrees C and that the protein conformation can be manipulated at elevated temperatures depending on solution conditions. The demonstrated conformational properties of these artificial proteins suggest that they may be excellent candidates for elucidating structure-function relationships in biopolymers for nanotechnology and biological applications.     

The end-to-end distance of RNA as a randomly self-paired polymer

In this paper, we present the end-to-end distance of randomly self-paired polymers (RSPPs). We define a randomly self-paired polymer as a linear polymer each of whose monomers has a probability, f(0<f<1), of pairing with any other one monomer. The RSPP model is inspired by numerous observations that the ends of RNAs are in close proximity. We use this model to explain this proximity. The prediction made by the RSPP model is consistent with these observations. Mapping an RNA with a length of 1000 nucleotides and a pairing fraction of 0.6 onto our RSPP model, for example, we predict an expected end-to-end distance of about 14 unpaired bases. We have also found that the expected end-to-end distance of the RSPP scales roughly as the 1/4 power of its total length.     

A brief review of coarse-grained and other computational studies of molecularly imprinted polymers

Molecular imprinting is an established method for the creation of artificial recognition sites in synthetic materials through polymerization and cross-linking in the presence of template molecules. Removal of the templates leaves cavities that are complementary to the template molecules in size, shape, and functionality. In recent years, various theoretical and computational models have been developed as tools to aid in the design of molecularly imprinted polymers (MIPs) or to provide insight into the features that determine MIP performance. These studies can be grouped into two general approaches-screening for possible functional monomers for particular templates and macromolecular models focusing on the structural characterization of the imprinted material. In this review, we pay special attention to coarse-grained models that characterize the functional heterogeneity in imprinted pores, but also cover recent advances in atomistic and first principle studies. We offer a critical assessment of the potential impact of the various models towards improving the state-of-the-art of molecular imprinting.     

Protein-based underwater adhesives and the prospects for their biotechnological production

Biotechnological approaches to practical production of biological protein-based adhesives have had limited success over the last several decades. Broader efforts to produce recombinant adhesive proteins may have been limited by early disappointments. More recent synthetic polymer approaches have successfully replicated some aspects of natural underwater adhesives. For example, synthetic polymers, inspired by mussels, containing the catecholic functional group of 3,4-L-dihydroxyphenylalanine adhere strongly to wet metal oxide surfaces. Synthetic complex coacervates inspired by the Sandcastle worm are water-borne adhesives that can be delivered underwater without dispersing. Synthetic approaches offer several advantages, including versatile chemistries and scalable production. In the future, more sophisticated mimetic adhesives may combine synthetic copolymers with recombinant or agriculture-derived proteins to better replicate the structural and functional organization of natural adhesives.     

Conversion of microbial rhodopsins: insights into functionally essential elements and rational protein engineering

Technological progress has enabled the successful application of functional conversion to a variety of biological molecules, such as nucleotides and proteins. Such studies have revealed the functionally essential elements of these engineered molecules, which are difficult to characterize at the level of an individual molecule. The functional conversion of biological molecules has also provided a strategy for their rational and atomistic design. The engineered molecules can be used in studies to improve our understanding of their biological functions and to develop protein-based tools. In this review, we introduce the functional conversion of membrane-embedded photoreceptive retinylidene proteins (also called rhodopsins) and discuss these proteins mainly on the basis of results obtained from our own studies. This information provides insights into the molecular mechanism of light-induced protein functions and their use in optogenetics, a technology which involves the use of light to control biological activities.     

Three approaches to assembling nano-bio-machines using molecular motors

Efforts to use protein molecular motors as nanoactuators are making rapid progress. For instance, it is now possible to carry out directional transport of small cargo along microtracks or microchannels using kinesin-microtubule systems, which could be the basis of micro-conveyor belts or molecular shuttles. However, the applicability of protein-based devices is limited by their poor stability in artificial environments. In addition, assembly of complex, intelligent microdevices or systems will likely require bottom-up self-assembly, and we still do not have sufficient knowledge to rationally design self-assembling protein-based microdevices or systems. One approach to solving the problems associated with protein-based systems is to use DNA-based nanodevices, which are amenable to rational design. Indeed, ingenious design has enabled realization of DNA-based nanoactuators and self-assembled micropatterns of various shapes. One also could use cells, organelles, or tissues as preassembled motile units, and several motile devices have already been realized using this approach. In addition to being less prone to the assemaly problems, cell-based microdevices have the advantage that the motile units reproduce themselves, and genetically encoded functional modifications can be replicated effortlessly. These protein-based, DNA-based, and cell-based systems each have distinct advantages and disadvantages, so that hybrid devices combining the best characteristics of all three would seem the most likely to succeed.     

Genetic engineering of self-assembled protein hydrogel based on elastin-like sequences with metal binding functionality

Recombinant DNA methods have been exploited to enable the creation of protein-based block copolymers with programmable sequences, desired properties, and predictable three-dimensional structures. These advantages over conventional polymer counterparts facilitate the utility of this new class of biomaterials in a wide range of applications. In this project, we exploited the environmental application of protein-based block copolymers based on elastin-like protein (ELP) sequences. Triblock copolymers containing charged and hydrophobic segments were synthesized. Chain lengths of each segment were manipulated in order to maintain a gelation point below room temperature. Polyhistidine sequences were successfully incorporated into the hydrophilic segment without disruption of the self-assembled hydrogel formation. The microscopic structure was further investigated using laser confocal microscopy. The metal binding capability and capacity of resulting hydrogel were studied to demonstrate the functionality of polyhistidine and its environmental application for heavy metal removal. Reversibility of metal binding was demonstrated, indicating the cost-effectiveness of this hydrogel. Significantly, we envision that this versatile strategy of incorporating functional groups within a 3-D protein network provides new possibilities in creation of biomaterials with great control over structure-property relationships.     

In silico Structure Modeling and Comparative Analysis of Characterization Properties of Protein Polymers Useful for Protein-Based Nano Particulate Drug Delivery Systems (NPDDS): A Bioinformatics Approach

With an increasing interest in nanoparticulate delivery systems, there is a greater need to identify biomaterials that are biocompatible and safe for human applications. Protein polymers from animal and plant sources are promising materials for designing nanocarriers. Composition of the protein plays an important role for specific drug delivery applications such as drug release, targeting, and stimuli responsive drug release. An important issue in protein polymers is characteristics such as size, charge, and hydrophobicity may play a significant role in phagocytic uptake and initiating a subsequent immune response. This remains to be investigated systematically by analyzing factors that influence nanoparticle characteristics of protein and reduce phagocytic uptake and does not initiate immune response too. Although protein polymers are biodegradable, it is essential to ensure that there must not be premature enzymatic breakdown of the protein nanoparticles in the systemic circulation. Surface modification of the protein nanoparticles can be used to address this issue to propose the necessary modification in the surface of the protein would be great contribution in the nano particulate drug delivery systems (NPPDS). Of the various proteins, gelatin and albumin have been widely studied for drug delivery applications. Plant proteins are yet to be investigated widely for drug delivery applications so there is need to find out the plant proteins capable to act as nanoparticles. The commercial success of albumin-based nanoparticles has created an interest in other proteins. An increased understanding of the physicochemical properties coupled with the developments in rDNA technology will open up new opportunities for protein-based nanoparticulate systems. In the present studies several proteins currently useful for drug delivery system were structurally modeled and has been analyzed to propose the essential characteristics of protein for protein-based NPDDS.     

Automated constraint-based nucleotide sequence selection for DNA computation

We present techniques for automating the design of computational systems built using DNA, given a set of high-level constraints on the desired behavior and performance of the system. We have developed a program called SCAN that exploits a previously implemented computational melting temperature primitive to search a 'nucleotide space' for sequences satisfying a pre-specified set of constraints, including hybridization discrimination, primer 5' end and 3' end stability, secondary structure reduction, and prevention of oligonucleotide dimer formation. The first version of SCAN utilized 24 h of computer time to search a space of over 7.5 billion unary counter designs and found only nine designs satisfying all of the pre-specified constraints. One of SCAN's designs has been implemented in the laboratory and has shown a marked improvement in performance over the products of previous attempts at manual design. We conclude with some novel ideas for improving the overall speed of the program that offer the promise of an efficient method for selecting optimal nucleotide sequences in an automated fashion.     

Antibody microarrays: current status and key technological advances

Antibody-based microarrays are among the novel classes of rapidly evolving proteomic technologies that holds great promise in biomedicine. Miniaturized microarrays (< 1 cm2) can be printed with thousands of individual antibodies carrying the desired specificities, and with biological sample (e.g., an entire proteome) added, virtually any specifically bound analytes can be detected. While consuming only minute amounts (< microL scale) of reagents, ultra- sensitive assays (zeptomol range) can readily be performed in a highly multiplexed manner. The microarray patterns generated can then be transformed into proteomic maps, or detailed molecular fingerprints, revealing the composition of the proteome. Thus, protein expression profiling and global proteome analysis using this tool will offer new opportunities for drug target and biomarker discovery, disease diagnostics, and insights into disease biology. Adopting the antibody microarray technology platform, several biomedical applications, ranging from focused assays to proteome-scale analysis will be rapidly emerging in the coming years. This review will discuss the current status of the antibody microarray technology focusing on recent technological advances and key issues in the process of evolving the methodology into a high-performing proteomic research tool.     

Tailored recombinant elastin-like polymers for advanced biomedical and nano(bio)technological applications

The genetic engineering of protein-based polymers is a method that enables, in an easy way, the design of complex and highly functional macromolecules. As examples of this approach, different molecular designs are presented, with increasing degree of complexity, showing how the controlled increase in their complexity yields (multi)functional materials with more selected and sophisticated properties. The simplest designs show interesting properties already, but the adequate introduction of given chemical functions along the polymer chain provides an opportunity to expand the range of properties to enhanced smart behavior and self-assembly. Finally, examples are given where those molecular designs further incorporate selected bioactivities in order to develop materials for the most cutting edge applications in biomedicine and nano(bio)technology.     

A plasmid display platform for the selection of peptides exhibiting a functional cell-penetrating phenotype

Cell-penetrating peptides (CPPs) represent a promising nonviral platform for the delivery of therapeutic cargos to cells and tissues. However, these peptides are often nonspecific, and their mechanism of action is still a subject of debate, which hinders the design of new CPPs. The alternative to rational protein design is the combinatorial approach to protein engineering, whereby large libraries of peptides are created and a screening or selection procedure is used to identify members with the desired phenotype(s). Here we describe a novel procedure for selecting peptides with a CPP phenotype using a plasmid display (PD) platform to link the peptides to their encoding DNA sequences. The PD system is based on genetic fusions to a DNA binding domain. The plasmid was designed to concomitantly express a fluorescent reporter protein to serve as a mock therapeutic cargo indicating its functional delivery into a cell. We have demonstrated this selection strategy using a control CPP (the TAT peptide) in the PC12 neuronal-like cell line. In the absence of transfection reagents, TAT was unable to deliver the protein/DNA complexes. The inclusion of the HA2 peptide from the hemagglutinin protein and the addition of polyethylenimine (PEI) were similarly ineffective. The addition of Lipofectamine, however, enabled the TAT-mediated delivery of the protein/DNA complexes, which was significantly better than control experiments without a CPP. This new PD selection platform will be a valuable new approach for use in identifying unique CPPs from randomized libraries with novel abilities and specificities.     

Adding functional entities to plasmids

Non-viral gene therapy constitutes an alternative to the more common use of viral-mediated gene transfer. Most gene transfer methods using naked DNA are based upon non-sequence-specific interactions between the nucleic acid and cationic lipids (lipoplex) or polymers (polyplex). We have developed a technology in which functional entities hybridize in a sequence-specific manner to the nucleic acid (bioplex). This technology is still in its infancy, but has the potential to become a useful tool, since it allows the construction of highly defined complexes containing a variety of functional entities. In its present form the bioplex technology is based upon the use of peptide/nucleic acids (PNA) as anchors. Single, or multiple, functional entities are directly coupled to the anchors. By designing plasmids, or oligonucleotides, with the corresponding anchor target sequence, complexes with desired composition can easily be generated. The long-term aim is to combine functional entities in order to achieve optimal, synergistic interactions allowing enhanced gene transfer in vivo.     

A multipurpose vector system for the screening of libraries in bacteria, insect and mammalian cells and expression in vivo

We have constructed a novel tetra-promoter vector (pBVboostFG) system that enables screening of gene/cDNA libraries for functional genomic studies. The vector enables an all-in-one strategy for gene expression in mammalian, bacterial and insect cells and is also suitable for direct use in vivo. Virus preparation is based on an improved mini Tn7 transpositional system allowing easy and fast production of recombinant baculoviruses with high diversity and negligible background. Cloning of the desired DNA fragments or libraries is based on the recombination system of bacteriophage lambda. As an example of the utility of the vector, genes or cDNAs of 18 different proteins were cloned into pBVboostFG and expressed in different hosts. As a proof-of-principle of using the vector for library screening, a chromophoric Thr⁶⁵-Tyr-Gly⁶⁷-stretch of enhanced green fluorescent protein was destroyed and subsequently restored by novel PCR strategy and library screening. The pBVboostFG enables screening of genome-wide libraries, thus making it an efficient new platform technology for functional genomics.     

Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system

We report a new strategy for the synthesis of genes encoding repetitive, protein-based polymers of specified sequence, chain length, and architecture. In this stepwise approach, which we term "recursive directional ligation" (RDL), short gene segments are seamlessly combined in tandem using recombinant DNA techniques. The resulting larger genes can then be recursively combined until a gene of a desired length is obtained. This approach is modular and can be used to combine genes encoding different polypeptide sequences. We used this method to synthesize three different libraries of elastin-like polypeptides (ELPs); each library encodes a unique ELP sequence with systematically varied molecular weights. We also combined two of these sequences to produce a block copolymer. Because the thermal properties of ELPs depend on their sequence and chain length, the synthesis of these polypeptides provides an example of the importance of precise control over these parameters that is afforded by RDL.