Proteins from thermophilic organisms generally show not only greater thermal stability, but also stability towards chemical denaturants and proteolysis (Vieille & Zeikus 2001, Leuschner & Antranikian 1995, Jaenicke 1991). Since not all enzymes are presently found in thermophiles and thermophilic proteins sometimes do not fold into the native conformation when expressed in E. coli, the overall stability of a target enzyme can be raised with directed evolution. However, it had previously been observed that the emergence of a desirable trait in an enzyme is accompanied by an evolutionary cost in some other properties, such as stability, activity or specificity (Antikainen et al. 2003, Celenza et al. 2008, Valderrama et al. 2002). Even though it is virtually impossible to predict these costs, it is widely believed that improvements in enzyme activity through protein engineering often come at the cost of reduced stability. For this reason, a stable enzyme was thought to evolve a function more efficiently than a similar, but less stable enzyme. This hypothesis has been demonstrated experimentally, by comparing the evolvability of marginally stable and thermostable variants of cytochrome P450 BM3 (Bloom et al. 2006). However, variants were only examined through one round of directed evolution. In general, directed evolution uses multiple generations of mutation and selection to improve or alter biochemical functions of proteins. In our study, we compare the evolvability of wild-type and thermostable variants of β-GUS for up to 5 rounds of directed evolution. The laboratory evolved version of the GUS-WT, that is GUS-TR3337, not only exhibited higher thermostability (Xiong et al. 2007) but also showed better resistance to chemical denaturants and mutations than wild type β-GUS. These comparative experiments show that, even though the stable GUS-TR3337 protein started with a lower glucosidase activity at room temperature relative to GUS-WT, the best variants obtained during each generation from GUS-TR3337 library achieved higher glucosidase activity and tolerance towards OTG than those from GUS-WT library.
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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.
A novel method to link a nascent protein (phenotype) to its mRNA (genotype) covalently through the N-terminus was developed. The mRNA harboring amber stop codon at just downstream of initiation site was hybridized with hydrazide-modified ssDNA at upstream of coding region and was ligated to the DNA. This construct was then modified with 4-acetyl-phenylalanyl amber suppressor tRNA. This modified construct was fused with the nascent protein via the phenylalanine derivative when the mRNA uses the amber suppressor tRNA to decode the amber stop codon. The obtained fusion molecule was used successfully in selective enrichment experiments. It will be applicable for high-through-put screening in evolutionary protein engineering. In contrast to fusion molecules generated by other methods in which the protein is linked to genotype molecule through the C- terminus, our fusion molecule will serve to select a protein for which the C-terminus is essential to be active. Key words: In vitro selection, mRNA-display, in vitro virus, C-terminus, Non-natural amino acid, T4 RNA Ligase
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We have shown that this multi-site incorporation method can utilize altered sets of 20 amino acids to design and engineer proteins and protein-like macromolecules. 9 By manipulation of both synthetase and tRNA in the host, one can also accomplish site- specific introduction of a single copy of a novel amino acid into proteins in vivo. 22-31 This method is derived from an in vitro approach to nonnatural amino acid incorporation through nonsense (stop codon) suppression, in which a stop codon (amber codon) was suppressed by a suppressor tRNA that had been chemically misacylated with the amino acid analog of interest. 32-36 Such a chemical method to alter the aminoacylation in vitro suffers from the technical difficulty and the intrinsic low yield of protein production, which limits its application. In 1998, Furter modified the cellular aminoacylation reaction by imparting a “twenty-first pair” comprising a yeast suppressor tRNA and a yeast phenylalanyl-tRNA synthetase ( y PheRS). 26 This approach allowed site-specific incorporation of L- p -fluorophenylalanine in vivo in response to an amber codon. More recently Schultz and colleagues have devised powerful selection methods to find useful mutant forms of tyrosyl-tRNA synthetase from archaebacterium Methanococcus jannaschii. 10,37 By introduction of such a mutant and a cognate suppressor, many
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To further assess specificity of VB15, we conducted far-western blotting analysis. NIH3T3, HEK293T and S2 cell lysates were probed using biotinylated VB15 as primary reagent. We observe a single bright band in the lane corresponding to NIH3T3 lysate indicating that VB15 specifically binds a single protein in NIH3T3 cells (Fig 3.6). We also observe a band at the same molecular weight in lanes corresponding to HEK293T and S2 lysates indicating that the target protein is conserved across species. However we see other bands, albeit not as bright in the HEK293T and S2 lanes indicating either the presence of other isoforms or some non specific binding. It is likely that since NIH3T3 cell homogenates were used for majority of the sorts it is the most specific in this case. However since the binding to the target appears to be conserved across species (mouse, human and drosophila) VB15 can still be used to IP vesicles in all three cell lines. A far western blot was performed using biotinylated Sso6904 gave no specific band for any of the lysates indicating that the specificity was engineered into VB15 during the sorting process.
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function 23 . Generally, these models are trained using large data sets composed of literature data from varied sources with little to no standardization of the experi- mental conditions, and trained using many protein classes (i.e. proteins with various folds and functions), because their aim is to identify sequence elements across all proteins that contribute to the property of interest. This generalist approach, how- ever, is not useful for identifying subtle sequence features (i.e. amino acids or amino acid interactions) that condition expression and localization for a specific class of related sequences, the ChRs in this case. We focused our model building on ChRs, with training data collected from a range of ChR sequences under standardized con- ditions. We applied Gaussian process (GP) classification and regression 24 to build models that predict ChR expression and localization directly from these data. In our previous work, GP models successfully predicted thermal stability, substrate binding affinity, and kinetics for several soluble enzymes 25 . Here, we asked whether GP modeling could accurately predict mammalian expression and localization for heterologous integral membrane ChRs and how much experimental data would be required. For a statistical model to make accurate predictions on a wide range of ChR sequences, it must be trained with a diverse set of ChR sequences 24 . We chose to generate a training set using chimeras produced by SCHEMA recombi- nation, which was previously demonstrated to be useful for producing large sets (libraries) of diverse, functional chimeric sequences from homologous parent pro- teins 26 . We synthesized and measured expression and localization for only a small subset (0.18%) of sequences from the ChR recombination library. Here we use these data to train GP classification and regression models to predict the expression and localization properties of diverse, untested ChR sequences. We first made pre- dictions on sequences within a large library of chimeric ChRs; we then expanded the predictions to sequences outside that set.
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[15, 16]. Notably, the codon-anticodon pairing is independent of the nature of the amino acid appended to the tRNA . Naturally many groups have focused on producing misacylated tRNA, which can then be accepted by the ribosome and allow the production any protein containing this amino acid. The Chamberlain and Schultz groups first reported the successful incorporation of unnatural amino acids using cell free translation systems in conjunction with chemically acylated suppressor tRNA (Figure 1.2) [18-21]. Subsequently it was shown that Xenopus oocytes, injected with chemically acylated suppressor tRNA and mRNA encoding a target gene with an internal suppression site, could synthesize a target protein bearing the unnatural amino acid site-specifically (Figure 1.3) . The target protein in these studies, nicotinic acetylcholine receptor (nAChR), is ideal because although the technique produces very little protein modern electrophysiology allows the detection of a very small number of active membrane ion channels, attomols of protein are sufficient (Figure 1.4). This system has allowed the elegant biophysical probing of structure/activity relationships of nAChR [24-27], but highlights the general caveats of chemical acylation for in vivo unnatural amino acid incorporation, production and delivery of chemically acylated tRNA and yield of the target protein.
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Fluorescent proteins have a tendency to dimerize at high concentrations. A crystal structure of GFP shows a hydrophobic dimer interface comprised of amino acid residues Ala206, Leu221, and Phe223 (Figure 4-11A) 37 . A strictly monomeric form of XFP can be obtained by mutating Ala206 to a Lys residue which introduces a long, positively charged side chain that disrupts the hydrophobic interface 38 . Fluorescent protein dimerization is likely to occur when restricted to two-dimensional space as when fused to membrane proteins 39 . To determine if XFP dimerization was having an effect on channel function or possibly even stoichiometry of GluCl, an A206K mutation was incorporated into the engineered constructs. The IVM concentration-response curve of the wild-type monomeric YFP-tagged (mYFP) receptor was no longer right-shifted compared to the WT receptor, and even revealed a distinctive second component (Figure 4-11B). Incorporation of mYFP into the ( α )L9’F receptor produced a more pronounced biphasic relationship than any previously observed. The same extreme biphasic behavior resulted when the L9’F mutation was present in the β subunit or present both α and β subunits (Figure 4-11C). Addition of ( β )Y182F to the ( α )L9’F mutation with mYFP tags now maintained a high sensitivity component, however the proportion was still reduced (Figure 4-11D).
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Since the advent o f solid phase peptide synthesis (SPPS) back in the early 60 s, a considerable amount o f effort has been directed towards the chemical synthesis of proteins. As has been described in the introduction, the use o f SPPS has been limited to molecules o f about 150 residues, due to the exponential accumulation of closely related impurities, which after 150 cycles represent a large proportion of the crude product and make purification to homogeneity difficult if not impossible. Furthermore, current methods for determining sequence homology are generally unable to offer definitive analysis o f the total protein sequence ie. the presence of a sequence lacking one amino acid would be difficult to detect. Hence fragment condensation may not only offer a route to improving SPPS for short proteins, but may also open an avenue towards the construction of sequences longer than 150 residues. Fragment condensation involves constructing short segments of the desired sequence which retain their side-chain protecting groups. These fully protected peptide fragments are then purified before being coupled together to give the complete peptide chain. The ability to fully characterise these shorter fragments would give far greater confidence to the homology of the final product than if the same protein had been synthesised in a stepwise fashion.
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One of the promises held out by protein engineering is the ability to predictably alter the properties of an enzyme to enable it to attack new substrates or catalyse existing substrates more efficiently. The promising aspect of such m anipulations is its im portance enzymologically and potentially industrially. One p articu larly well characterised enzyme which is suited for protein engineering is the alcohol dehydrogenase as its complete amino acid sequence is known from a range of organisms and its crystal stru ctu re has been solved for horse ADH. Inform ation on presum ed enzyme m echanism s has been gained from com parative studies on homologous enzymes by relating the observed amino acid substitutions to changes in function. Such studies have indicated th a t yeast and m am m alian alcohol dehydrogenases are homologous (Jornvall et al.y 1978) b u t they differ in q u a rte rn a ry stru ctu re and su b stra te
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significant differences in secondary structure and unfolding behavior as a function of temperature. These results can be seen in figure 1.6. These interesting experiments suggest that the hydrophobicity of the moieties present at positions in the rhPrP C normally occupied by methionine can significantly impact how well the protein maintains its folded state, and that hydrophilic moieties appear to substantially impact the proper folding of the protein in question. Because methionine oxidation results in the formation of more hydrophilic functionalities at methionine positions, this work suggests that oxidative stresses may play a role in the development of prion disease and other diseases caused by protein misfolding. These techniques may also be applicable to studying a number of other cellular proteins that can undergo oxidation at methionine residues (195-197). Studying protein stability using global perturbations introduced with ncAAs appears to be useful in a variety of settings. Both model proteins and more complex proteins can be perturbed in ways such that protein stability is either negatively or positively affected. Observing these changes in stability provides fundamental information regarding how changing molecular properties results in changes to protein properties as a whole and also provides ideas for engineering proteins with altered stabilities. Thus, global perturbations provide a complementary approach to local, site-specific perturbations. These two approaches add substantial capability to researchers’ toolkit for assessing factors contributing to protein stability and engineering more stable, faster folding proteins.
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Being protein function a conformation-dependent issue, avoiding aggregation during production is a major challenge in biotechnological processes, what is often successfully addressed by convenient upstream, midstream or downstream approaches. Even when obtained in soluble forms, proteins tend to aggregate, especially if stored and manipulated at high concentrations, as is the case of protein drugs for human therapy. Post-production protein aggregation is then a major concern in the pharmaceutical industry, as protein stability, pharmacokinetics, bioavailability, immunogenicity and side effects are largely dependent on the extent of aggregates formation. Apart from acting at the formulation level, the recombinant nature of protein drugs allows intervening at upstream stages through protein engineering, to produce analogue protein versions with higher stability and enhanced therapeutic values.
Abstract: Chiral amines are important components of 40–45% of small molecule pharmaceuticals and many other industrially important fine chemicals and agrochemicals. Recent advances in synthetic applications of ω -transaminases for the production of chiral amines are reviewed herein. Although a new pool of potential ω-transaminases is being continuously screened and characterized from various microbial strains, their industrial application is limited by factors such as disfavored reaction equilibrium, poor substrate scope, and product inhibition. We present a closer look at recent developments in overcoming these challenges by various reaction engineering approaches. Furthermore, protein engineering techniques, which play a crucial role in improving the substrate scope of these biocatalysts and their operational stability, are also presented. Last, the incorporation of ω -transaminases in multi-enzymatic cascades, which significantly improves their synthetic applicability in the synthesis of complex chemical compounds, is detailed. This analysis of recent advances shows that ω -transaminases will continue to provide an efficient alternative to conventional catalysis for the synthesis of enantiomerically pure amines.
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SSP_Lrp gene, outside the SP biosynthetic gene clus- ter, was identified from the genomic DNA sequence of S. spiramyceticus 1941, as a global regulator involved in the regulation of BT or SP biosynthesis. In this study, we showed that SSP_Lrp protein specifically bound to the promoter regions of acyB2, bsm23, and bsm42 genes which are positive regulatory genes located in BT bio- synthetic gene cluster. Therefore, we assumed that the SSP_Lrp regulator was a higher hierarchy member in reg- ulatory networks of SP and BT biosynthesis. But the SP production of SSP_Lrp-null mutant (ΔSSP_Lrp) was just improved a little and with similar phenotype to wild type. However, high expression of SSP_Lrp in S. spiramyceti- cus 1941 significantly decreased the yield of SP. By delet- ing the l-Leu putative binding domain of SSP_Lrp in S. spiramyceticus 1941, the production of SP and BT were evidently increased, furthermore, significantly improved on the SP III and ISP III components. These results dem- onstrated that SSP_Lrp played a negative role involved in SP or BT biosynthesis. It may depress the transcription of positive regulatory genes or lessen the precursor supply of SP biosynthesis.
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Another important aspect of the Chapter 3 work is the selection of functional dimeric ligands which form on the surface of yeast. A hypothesis around this observation is that prior to being exported to the yeast surface M2 ligands self-associate and form an intermolecular-disulfide bond. This results in co-localization of the two dimerized M2 ligands on the surface of yeast. The same co- localization of ligands likely does not occur without the driving force of self-affinity. Accordingly, determining if yeast displayed ligands are co-localized should enable the selection of monomeric or dimeric ligands. It would be useful to screen for dimerization as this would remove the trial and error approach currently used to determine if a ligand is monomeric or dimeric. The current technique is to clone the yeast displayed ligand into an expression vector, express and purify the mutant and then evaluate the oligomeric state in a size exclusion column. One method to screen for co-localization of displayed ligands would be to develop a molecular ruler which would act as a biosensor. The biosensor would be comprised of two identical and linked scaffold derived ligands targeted to the HA-tag. The HA-tag ligands would need to be selected to have low affinity when only monovalent binding occurs but bind with high affinity when the two linked ligands bind distinct HA tags. This bivalent protein could be fused to a monomeric fluorescent protein such as TagGFP. FACS could then be used to select for GFP positive or negative yeast cells of interest. Additional examples of dimeric and monomeric ligands selected by yeast surface display exist in the literature and could serve as additional positive and negative controls to validate the tool 13,15 . Figure 4.1 illustrates the
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The screening of a large number of gene knock-in and knock-outs to select the desirable phenotype of improved expression capability is time consuming. The simplest ap- proach to screen a very large number of clones is to use FACS based screening for cells expressing fluorescence tagged proteins like GFP [179,180]. Thus libraries with engineered genomes can be screened for the highest pro- ducers by using appropriate sorting protocols [181,182]. However such modified hosts may not necessarily over express other proteins, given the very specific nature of host-protein interactions. Another strategy would be the selection of quiescent phenotype, in order to uncouple growth and product formation. For this one can screen for a growth stoppage phenotype which typically leads to elongated cell morphologies due to stoppage of cell div- ision . Simultaneously or later these cells can be checked for recombinant protein expression capability after growth arrest. Such techniques can be coupled with auto- mated devices where cultures can grow in 96 well plate for- mats. Such technologies have proven to work well in clone screening and help in quickly identifying the best per- formers from a large number of clones e.g. BioLector from m2p Labs [184,185], Bioscreen C from Oy Growth Curves Ab Ltd [186-190], Clone Screener from Biospectra AG and the Ambr reactor from TAPBiosystems. Apart from growth profiling, these systems can also do online monitoring of fluorescence, pH, dissolved oxygen and NADH  and are reviewed in [191-195].
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Importantly, the unfolded protein response and the master lipid flux regulatory role of Pah1p orthologues (lipins) , are conserved in all eukaryotes, including insect cells and mammalian cells. Technology to knock down or knock out genes in these organisms is now readily available [22,23]. Therefore, there is scope for applying the same membrane engineering approach to these other frequently used (membrane) protein expres- sion hosts. However, careful exploration of an appropri- ate match between the carbon source and the promoter systems will be required for other biotech cell types in which the Δpah1 manipulation is attempted and this will be the subject of further studies. For example, while Pichia pastoris is widely used for heterologous protein expression and extremely strong methanol-inducible promoters are available, our preliminary data (unpub- lished) suggest that C1-metabolism is not compatible with Δpah1-mediated membrane expansion: the mem- branes appear to be autophagocytosed, obliterating any beneficial effect on membrane protein expression yields. However, the rapidly expanding genetic toolbox for Pichia  will allow to match appropriate promoter
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T he nonautonomous adeno-associated virus (AAV) is not associated with disease, and thus there has historically been less incentive to study basic AAV biology in as great depth as that of human pathogens. Clinical success with AAV-based gene therapy vectors have elevated the interest in the study of AAV biology to inform vector engineering efforts. Within the family Parvoviridae, AAV represents one of the smallest and simplest mammalian viruses, AAV is a nonenveloped, 25-nm, T ⫽ 1 icosahedral capsid containing only the 4.7-kb genome. AAV The single-stranded DNA (ssDNA) genome includes two genes, rep, which encodes four nonstructural proteins, and cap, which encodes the three structural proteins (VP). The different protein products are generated through alternative splicing (1) and noncanonical translation start codons (2, 3), such that the products of the respective gene share portions of overlapping identity yet remain functionally distinct. For example, VP3 is ⬃ 530 amino acids in length and takes on a characteristic fold that forms the basic structural capsid monomer (4); VP2 and VP1 contain the entire VP3 sequence and same structural fold, but extend N-terminally by ⬃ 65 and ⬃ 202 residues, respectively. These N-terminal “tails” are disordered and situated in the interior of a newly assembled capsid (5); during the entry phase of infection, the VP1 tail is extruded (6) through a pore at the 5-fold axis of symmetry (7). This VP1 unique region contains a phospholipase domain critical for infectivity (8), whereas VP2 has been demonstrated to be dispensable for capsid assembly and virion function (9).
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The ECM can serve many functions, such as providing support, segregating tissues from one another, and regulating intercellular communication. The extracellular matrix regulates a cell's dynamic behaviour. In addition, it sequesters a wide range of cellular growth factors and acts as a local depot for them. Formation of the extracellular matrix is essential for processes like growth, wound healing and fibrosis. Fibrosis is the formation of excess fibrous connective tissue in an organ or tissue in a reparative or reactive process. Fibrosis can be used to describe the pathological state of excess deposition of fibrous tissue, as well as the process of connective tissue deposition in healing. Scaffolds are artificial structures that are capable of supporting 3D tissue formation. The cells are implanted or seeded in scaffolds and are critical both ex vivo as well as in vivo. The terms ex- vivo and in vitro are not synonymous. In vivo studies are those that are conducted with living organisms in their normal intact state. Ex vivo studies are conducted on functional organs that have been removed from the intact organism. In vitro studies are conducted using components of an organism that have been isolated from their usual biological surroundings. They are commonly called as test tube experiments. In- vitro means ‘in glass’ in Latin. To restore function or regenerate tissue, a scaffold is necessary that will act as a temporary matrix for cell proliferation and extracellular matrix deposition, with subsequent ingrowth until the tissues are totally restored or regenerated. Scaffolds have been used for tissue engineering such as bone, cartilage, ligament, skin, vascular tissues, neural tissues, and skeletal muscle and as vehicle for the controlled delivery of drugs, proteins, and DNA .
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Synthetic biology promises to revolutionize biotechnology by enabling the rational design of genetic constructs (parts) and cells (chassis) with predictable user-defined functions. It is particularly applicable to recombinant protein manufacturing, where the engineered system comprises just two key biological components, a recombinant gene expression cassette and a host- cell factory, and two critical system outputs, product yield and quality. Although diverse eukaryotic and prokaryotic cell-types are used to produce recombinant proteins, the core biomanufacturing process remains constant. A cell factory must create and maintain cellular biosynthetic capacity, and utilize it to synthesize a complex protein product. As shown in Figure 1, product yields are a function of the rates of five key biosynthetic steps (four if product membrane translocation is not required), each of which can be controlled by re-design of either the cell chassis or a discrete genetic component. Strategies to enhance product yield have traditionally been limited to increasing the output from a single one of these steps, typically resulting in relatively modest improvements (Davy et al., 2017; Wells & Robinson, 2017; Xiao et al., 2014). Given recent advances in DNA-part and chassis engineering, we should be able to move beyond modulation of individual cellular processes, and create a new paradigm for biomanufacturing where the entire product biosynthetic pathway is specifically designed to maximize system output.
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