Chlorinated amino acids in peptide production

A new method for the production of peptides through biological expression was developed, utilising lactonisation-prone chlorinated amino acids for latent peptide bond digestion. Incorporation of halogenated amino acids into proteins is possible due to the inherent inability of the biological synthetic machinery to discriminate against compounds structurally similar to the natural substrates. As isoleucine, leucine and valine are primarily recognised by size exclusion, all isosteres with a methyl group replaced by similarly-sized chlorine are mistaken as substrates, and the ability of halogens to mimic the bonding interactions of sulfur enabled the design of a chlorinated and a brominated analogue of methionine. Amino acids with chlorine at the 4-position, incorporated into proteins in place of isoleucine, leucine or methionine during cell-free protein expression, were found to trigger cleavage of the proximal peptide bond on the C-terminal side at elevated temperatures. The reaction mechanism is similar to that of cyanogen bromide induced cleavage, driven by the formation of a highly favourable 5-membered ring. This presents the first case of heat induced proteolysis, resulting in almost instantaneous peptide bond cleavage through exposure to 100 degrees Celsius in water, and avoids the need for toxic, expensive or sensitive external agents. When encoded in a fusion protein, the chlorinated residues can be used for rapid separation of the fusion partners. The utility of the method was demonstrated through the preparation of small peptides human gastrin releasing peptide prohormone, cholecystokinin prohormone and oxytocin, lacking isoleucine, leucine and methionine, respectively, expressed as fusion proteins. Through simultaneous replacement of two amino acids, deuterated and fluorinated analogues of the peptides were also prepared. The method was then expanded to prepare more complicated targets. Homologous substitution of leucine with isoleucine, and vice versa, enabled the preparation of a small protein aprotinin. A crystal structure of the mutated aprotinin demonstrated that the protein structure was not affected by the substitution, thus establishing that interchange of similar amino acids can be used to overcome sequence limitations. In stark contrast, amino acids with chlorine at the 3-position are resistant to high temperature. Through the ability to substitute valine or isoleucine during protein expression, the chlorides were incorporated into aprotinin, thus establishing a method for the preparation of proteins of essentially unlimited size containing 3-chloro amino acids. Crystal structures of the chlorinated aprotinin showed that the amino acid analogues are not inherently detrimental to protein structure and can lead to stable proteins, while the preparation through the expression of heat-labile fusion proteins juxtaposed the differences in reactivity between amino acids halogenated at the 3- and 4-positions

Protein-directed crystalline 2D fullerene assemblies

Water soluble 2D crystalline monolayers of fullerenes grow on planar assemblies of engineered consensus tetratricopeptide repeat proteins. Designed fullerene-coordinating tyrosine clamps on the protein introduce specific fullerene binding sites, which facilitate fullerene nucleation. Through reciprocal interactions between the components, the hybrid material assembles into two-dimensional 2 nm thick structures with crystalline order, that conduct photo-generated charges. Thus, the protein-fullerene hybrid material is a demonstration of the developments toward functional materials with protein-based precision control of functional elements

Designing artificial fluorescent proteins: Squaraine-LmrR biophosphors for high performance deep-red biohybrid light-emitting diodes

Biophosphors with fluorescent proteins (FPs) are promising candidates to replace rare-earth color down-converting filters for white light-emitting diodes (LEDs). There is, however, a lack of deep-red FPs meeting high photostabilities, photoluminescence quantum yields (ϕ), and throughput expression yields. Herein, a new approach for the design of highly emissive and stable deep-red biophosphors combining an artificial FP (Lactococcal multidrug resistance Regulator (LmrR) as protein host and an archetypal red-emitting squaraine (S) as guest) with a polymer network is demonstrated toward high performing deep-red biohybrid LEDs (Bio-HLEDs). At first, the best protein pocket (aromaticity, polarity, charge, etc.) to stabilize S in water is determined using four LmrR variants (position 96 with tryptophan, histidine, phenylalanine, and alanine). Computational and time-resolved spectroscopic findings suggest that the tryptophan is instrumental toward achieving artificial red-emitting FPs with ϕ > 50% stable over weeks. These features are further enhanced in the polymer coating (ϕ > 65% stable over months) without affecting emission color. Finally, deep-red Bio-HLEDs are fabricated featuring external quantum efficiencies of 7% and stabilities of ≈800 h. This represents threefold enhancement compared to reference devices with S-polymer color filters. Overall, this work highlights a new design for highly emissive deep-red biophosphors, achieving record performance in deep-red protein-LEDs.The authors acknowledge the European Union's Horizon 2020 research and innovation FET-OPEN under grant agreement ARTIBLED No. 863170. R.D.C. acknowledges the ERC-Co InOutBioLight No. 816856. P.B.C. acknowledges financial support from the Ministry of Science, Innovation and Universities of Spain under the Beatriz Galindo Programme (No. MCIU-19-BEAGAL 18/0224), from MCIN/AEI/10.13039/501100011033 grant No. PGC2018-095953-B-I00 and from the Technical University of Munich (TUM) under the TUM Global Visiting Professor Programme. A.L.C. acknowledges support by the European Research Council ERC-CoG-648071-ProNANO and ERC-PoC-841063-NIMM; and Agencia Estatal de Investigación, Spain (No. PID2019-111649RB-I00). This work was performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency Grant No. MDM-2017-0720 (CIC biomaGUNE).Peer reviewe

Fed-batch pre-industrial production and purification of a consensus tetratricopeptide repeat (CTPR) scaffold as a container for Fluorescent Proteins (FPs)

White light-emitting diodes (WLEDs) are big news in the field of lighting, however, current production processes are still very expensive or based on unsustainable inorganic metals such as inorganic phosphorus. The EU-funded ARTIBLED project aims to produce low-cost and high-efficiency Bio-hybrid light-emitting diodes (Bio-HLEDs). This can be achieved using artificially synthesized fluorescent proteins linked in biological scaffolds like the packaging to obtain LED for lighting applications containing a biogenic phosphor. This study aims to optimize the protein scaffold CTPR10 production process to obtain a high number of scaffolds with a good purity level for Bio-HLEDs construction. Different fed-batch fermentation procedures were investigated and it was possible to produce more than two times of biomass and intracellular proteins compared to a conventional fed-batch strategy. The improvement in production leads both to the reduction of costs related to the amount of IPTG used and the isolation of a consistent amount of CTPR10 through rapid and highly efficient purification techniques. The realization of this project represents a significant milestone for Europe which will be at the forefront of innovation in the lighting sector

In situ deprotection and incorporation of unnatural amino acids during cell-free protein synthesis

The S30 extract from E. coli BL21 Star (DE3) used for cell-free protein synthesis removes a wide range of α-amino acid protecting groups by cleaving α-carboxyl hydrazides; methyl, benzyl, tert-butyl, and adamantyl esters; tert-butyl and adamantyl carbo

Protein-Directed Crystalline 2D Fullerene Assemblies

Repeat proteins with engineered tyrosine clamps provide a platform for fullerene assembly into 2D crystalline materials with long range molecular order and photogenerated charge carrier capacity. Thus, the self-assembling hybrid material allows to utilise the innate properties of fullerenes, demonstrating the potential of engineered protein-based functional materials