Recombinant Limbs Assay to Understand Morphogenesis, Patterning, and Early Steps in Cell Differentiation

Understanding the early steps of cell differentiation is essential to know other complex processes such as morphogenesis. Thus, recombinant chicken limbs are an experimental model that allows the study of cell differentiation and pattern generation under embryonic patterning signals.

This experimental model imitates an in vivo environment; it assembles reaggregated cells into an ectodermal cover obtained from an early limb bud. Later, ectoderms are transferred and implanted in a chick embryo receptor to allow its development joplink Recombinant Human C5a anaphylatoxin. This assay was mainly used to evaluate mesodermal limb bud cells; however, it can be applied to other stem or progenitor cells from other organisms.

  • Organism Recombinants

Epigenomic and structural events preclude recombination in Brassica napus

-Meiotic recombination is a major evolutionary process generating genetic diversity at each generation in sexual organisms. However, this process is highly regulated with the majority of crossovers lying in the distal chromosomal regions that harbor low DNA methylation levels. Even in these regions, some islands without recombination remain, for which we investigated the underlying causes. -Genetic maps were established in two Brassica napus hybrids to detect the presence of such large non-recombinant islands.
  • The role played by DNA methylation and structural variations in this local absence of recombination was determined by performing BS-seq and whole genome comparisons. Inferred structural variations were validated using either optical mapping or oligo-FISH.
  • Hypermethylated or inverted regions between Brassica genomes were associated with the absence of recombination. Pairwise comparisons of nine B. napus genome assemblies revealed that such inversions occur frequently and may contain key agronomic genes such as resistance to biotic stresses.
  • We conclude that such islands without recombination can have different origins, such as DNA methylation or structural variations in B. napus. It is thus essential to take into account these features in breeding programs as they may hamper the efficient combination of favorable alleles in elite varieties.

Recombinant Protein Production and Purification of Insoluble Proteins

Proteins are synthesized in heterologous systems because of the impossibility to obtain satisfactory yields from natural sources. The efficient production of soluble and functional recombinant proteins is among the main goals in the biotechnological field. In this context, it is important to point out that under stress conditions, protein folding machinery is saturated and this promotes protein misfolding and, consequently, protein aggregation.
Thus, the selection of the optimal expression organism and its growth conditions to minimize the formation of insoluble protein aggregates should be done according to the protein characteristics and downstream requirements. Escherichia coli is the most popular recombinant protein expression system despite the great development achieved so far by eukaryotic expression systems. Besides, other prokaryotic expression systems, such as lactic acid bacteria and psychrophilic bacteria, are gaining interest in this field.
However, it is worth mentioning that prokaryotic expression system poses, in many cases, severe restrictions for a successful heterologous protein production. Thus, eukaryotic systems such as mammalian cells, insect cells, yeast, filamentous fungus, and microalgae are an interesting alternative for the production of these difficult-to-express proteins.

A Novel Potent Carrier for Unconventional Protein Export in Ustilago maydis

Recombinant proteins are ubiquitously applied in fields like research, pharma, diagnostics or the chemical industry. To provide the full range of useful proteins, novel expression hosts need to be established for proteins that are not sufficiently produced by the standard platform organisms. Unconventional secretion in the fungal model Ustilago maydis is an attractive novel option for export of heterologous proteins without N-glycosylation using chitinase Cts1 as a carrier. Recently, a novel factor essential for unconventional Cts1 secretion termed Jps1 was identified.
Here, we show that Jps1 is unconventionally secreted using a fusion to bacterial β-glucuronidase as an established reporter. Interestingly, the experiment also demonstrates that the protein functions as an alternative carrier for heterologous proteins, showing about 2-fold higher reporter activity than the Cts1 fusion in the supernatant.
In addition, Jps1-mediated secretion even allowed for efficient export of functional firefly luciferase as a novel secretion target which could not be achieved with Cts1. As an application for a relevant pharmaceutical target, export of functional bi-specific synthetic nanobodies directed against the SARS-CoV2 spike protein was demonstrated. The establishment of an alternative efficient carrier thus constitutes an excellent expansion of the existing secretion platform.

Recent advances in molecular farming using monocot plants

Heterologous synthesis of proteins or peptides in plant-based systems, referred to as plant molecular farming, is a practical and safe approach for the large-scale and cost-effective production of therapeutic biomolecules. In this context, monocotyledonous plants, and especially cereals, have been considered attractive vehicles for producing high-value recombinant proteins. The endosperm, as the largest grain storage compartment, offers an appropriate environment for long-lasting protein accumulation.
During the last decades, fascinating progress has been achieved in the gene transfer technology and genetic manipulation of the monocot crops using either Agrobacterium tumefaciens or direct gene transfer by biolistic methods. Our group has recently expressed biologically active recombinant human peptide cathelicidin in barley grains using endosperm-specific promoter and brought such engineered lines to field cultivation under current EU regulations for genetically modified organisms.
This article reviews the most recent advances and strategies for the production of biopharmaceutical proteins in transgenic monocots, highlighting various aspects involved in recombinant protein accumulation in grains, and discussing current bottlenecks and perspectives for the biosynthesis of therapeutic molecules using different monocot plant platforms.
The spirochete Leptospira interrogans serovar Copenhageni harbors the genetic elements of the CRISPR-Cas type I-B system in its genome. CRISPR-Cas is a CRISPR RNA (crRNA) mediated adaptive immune system in most prokaryotes against mobile genetic elements (MGEs). To eliminate the intruding MGEs, CRISPR-Cas type I systems utilize a Cascade (CRISPR-associated complex for antiviral defense) complex composed of Cas5, Cas6, Cas7, and Cas8 bound with a crRNA. The Cas7 is essentially known to constitute the major component of the Cascade complex. The present study reports the biochemical characterization of the Cas7 (LinCas7) from the CRISPR-Cas type I-B system of L. interrogans serovar Copenhageni.
The pure recombinant LinCas7 (rLinCas7) exists as a monomer in the solution by size exclusion chromatography. The rLinCas7 demonstrates an endoDNase activity dependent upon divalent Mg2+ ions, monovalent ions, pH, temperature, and substrate size. Analysis of ribonucleoprotein composite (rLinCas7-crRNA) by electron microscopy and native-PAGE demonstrated that rLinCas7 could oligomerize on the mature CRISPR RNA (crRNA) framework in the presence of Mg2+ ions.
The ribonucleoprotein composite attains a helical shape similar to the backbone of the Cascade complex. However, in the absence of Mg2+ ions, rLinCas7 acts as an RNase. The fluorescence spectroscopy disclosed a weak interaction (Kd = 26.81 mM) between rLinCas7 and Mg2+ ions, leading to an overall conformational change in rLinCas7 that modulates the rLinCas7’s activity on DNA and RNA substrates. The nuclease activity of LinCas7 characterized in this study aids to the functional divergences among proteins of the Cas7 family from different CRISPR-Cas systems in various organisms.

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