High throughput automated microbial bioreactor system used for clone selection and rapid scale-down process optimization

High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed-batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled-up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale-u

Copper-free sonogashira cross-coupling for functionalization of alkyne-encoded proteins in aqueous medium and in bacterial cells

Bioorthogonal reactions suitable for functionalization of genetically or metabolically encoded alkynes, for example, copper-catalyzed azide-alkyne cycloaddition reaction ("click chemistry"), have provided chemical tools to study biomolecular dynamics and function in living systems. Despite its prominence in organic synthesis, copper-free Sonogashira cross-coupling reaction suitable for biological applications has not been reported. In this work, we report the discovery of a robust aminopyrimidine-palladium(II) complex for copper-free Sonogashira cross-coupling that enables selective functionalization of a homopropargylglycine (HPG)-encoded ubiquitin protein in aqueous medium. A wide range of aromatic groups including fluorophores and fluorinated aromatic compounds can be readily introduced into the HPG-containing ubiquitin under mild conditions with good to excellent yields. The suitability of this reaction for functionalization of HPG-encoded ubiquitin in Escherichia coli was also demonstrated. The high efficiency of this new catalytic system should greatly enhance the utility of Sonogashira cross-coupling in bioorthogonal chemistry

Connecting biochemistry and elelctronics with artifical allosteric protein biosensors

The idea that biological systems can be built from standard components is the central tenet of Synthetic Biology. Although proteins control most of real time information and energy flow in a cell, our ability to create protein-based switches and thereon based circuits is woefully underdeveloped. We addressed this by developing a generally applicable strategy for converting constitutively active enzymes into allosterically controlled switches. This was achieved by constructing chimeric enzymes where the enzymatically active part is fused with a conformational switch. We demonstrated that such basic signaling units could be compiled into higher order biosensor systems capable of detecting potentially any analyte. Specifically using this approach we constructed electrochemical and optical biosensors specific to ions, small molecule drugs and proteins

Enhancing the thermal stability of a single-chain Fv fragment by in vivo global fluorination of the proline residues

Single-chain Fv (scFv) format protein is a commonly used analytical tool for diagnostic and therapeutic applications. The usage of scFv antibody fragments in therapeutic applications has demonstrated that they need to have high thermostability. Many rational or irrational methods have been described erstwhile to engineer or improve the stability of scFv proteins by modifications of natural amino acid. Here we have demonstrated an alternate strategy to efficiently improve the thermostability of scFvs by non-canonical amino acid. Previously, fluoroprolines have been proven as a choice to tune the stability of many polypeptides and few globular proteins. Hence we exploited the usage of fluoroproline to enhance the thermal stability of scFv by replacing the natural proline on the framework regions of scFv that influence the folding or stability. To demonstrate our approach, a bacterial cytoplasmic foldable and humanized anti-c-Met scFv (hu-MscFv) was used. The hu-MscFv proline sites were successfully incorporated with (2S,4R)-4-fluoroproline without affecting its structure and function by the in vivo residue specific global replacement method which exploits bacterial auxotrophic system. The time-dependent temperature effect on the activity of fluorinated hu-MscFv revealed its increased stability at 40 °C along with improved half-life than the hu-MscFv with natural proline. Further model based structure analysis on hu-MscFv with fluoroproline indicated that the fluorine atoms were able to establish new favourable dipole interactions with neighbouring polar groups in their local micro environments that rationalizes its improved thermostability. Moreover the scFv sequence based statistical analysis strongly supports the fact that this method can be applied to any target scFv, since they contain high frequency conserved proline sites in their framework regions

Control of Allosteric Protein Electrochemical Switches with Biomolecular and Electronic Signals

The construction of allosteric protein switches is a key goal of synthetic biology. Such switches can be compiled into signaling systems mimicking information and energy processing systems of living organisms. Here we demonstrate construction of a biocatalytic electrode functionalized with a recombinant chimeric protein between pyrroloquinoline quinone-dependent glucose dehydrogenase and calmodulin. This electrode could be activated by calmodulin-binding peptide and showed a high bioelectrocatalytic current (ca. 300 μA) due to efficient direct electron transfer. In order to expand the types of inputs that can be used to activate the developed electrode, we constructed a caged version of calmodulin-binding peptide that could be proteolytically uncaged using a protease of choice. Finally, the complexity of the switchable bioelectrochemical system was further increased by the use of almost any kind of molecule/biomolecule or electronic signal, unequivocally proving the orthogonality of the aforementioned system

Generation of efficient fingerprint for GFP-like fold and computational identification of potential GFP-like homologs

There is a considerable interest in the detection of GFP-like proteins due to their structural stability and functional usefulness. GFP-like proteins share highly conserved beta-barrel fold with 11 beta-strands. However, their low sequence identity hampers efficient identification of their homologous proteins from database. In this study, an attempt was made to generate a fingerprint for efficient detection of GFP-like proteins. Overlapped conserved residues (OCR)-based approach has been used to design a protein fingerprint based on sequentially and structurally conserved residues in secondary structures to detect homologous proteins very efficiently. Therefore, a fingerprint for GFP-like fold was designed using the OCR approach. However, its specificity was too low to be used for the identification of novel proteins. The conserved residues of loop regions were added and optimized to improve its specificity without losing its high sensitivity. The optimized fingerprint was employed to scan NR database. A total of 20 hypothetical proteins were detected, among which nine were validated as potential GFP-like homologs

Control of allosteric electrochemical protein switch using magnetic signals

We report a novel approach for magneto-controlled activation of an artificial electro-enzymatic cascade. The input signal triggers release of a caged ligand peptide, its proteolytic processing and activation of an artificial allosteric enzyme based on PQQ-dependent glucose dehydrogenase. The developed cascade was used to assemble a magneto-controlled biofuel cell

Control of allosteric electrochemical protein switch using magnetic signals

We report a novel approach for magneto-controlled activation of an artificial electro-enzymatic cascade. The input signal triggers release of a caged ligand peptide, its proteolytic processing and activation of an artificial allosteric enzyme based on PQQ-dependent glucose dehydrogenase. The developed cascade was used to assemble a magneto-controlled biofuel cell

Control of Allosteric Protein Electrochemical Switches with Biomolecular and Electronic Signals

The construction of allosteric protein switches is a key goal of synthetic biology. Such switches can be compiled into signaling systems mimicking information and energy processing systems of living organisms. Here we demonstrate construction of a biocatalytic electrode functionalized with a recombinant chimeric protein between pyrroloquinoline quinone-dependent glucose dehydrogenase and calmodulin. This electrode could be activated by calmodulin-binding peptide and showed a high bioelectrocatalytic current (ca. 300 μA) due to efficient direct electron transfer. In order to expand the types of inputs that can be used to activate the developed electrode, we constructed a caged version of calmodulin-binding peptide that could be proteolytically uncaged using a protease of choice. Finally, the complexity of the switchable bioelectrochemical system was further increased by the use of almost any kind of molecule/biomolecule or electronic signal, unequivocally proving the orthogonality of the aforementioned system