Dataset Information

Construction of E. coli-Mycobacterium shuttle vectors with a variety of expression systems and polypeptide tags for gene expression in mycobacteria.

ABSTRACT: Cloning and expression of a desired gene is indispensable in molecular biology studies. Expression vectors, in this regard, should offer much needed flexibility and choice of cloning strategies for both in vivo and in vitro protein expression experiments. Furthermore, availability of option to choose from various reporter tags allows one to be flexible during designing of an experiment in a more relevant manner. Thus, the need of a versatile expression system cannot be ignored. Although several different expression vectors are available for gene expression in mycobacteria, they lack the required versatility of expression and the inclusion of reporter tags. We here present the construction of a set of nine E. coli-Mycobacterium shuttle plasmids, which offer a combination of three mycobacterial promoter systems (heat shock inducible-hsp60, tetracycline-, and acetamide-inducible) along with three polypeptide tags (Green Fluorescent Protein (GFP), Glutathione S-transferase (GST) and hexa-histidine tag). These vectors offer the cloning of a target gene in all the nine given vectors in parallel, thus allowing the generation of recombinant plasmids that will express the target gene from different promoters with different tags. Here, while the hexa-histidine and GST tags can be used for protein purification and pull-down experiments, the GFP-tag can be used for protein localization within the cell. Additionally, the vectors also offer the choice of positioning of the reporter tag either at the N-terminus or at the C-terminus of the expressed protein, which is achieved by cloning of the gene at any of the two blunt-end restriction enzyme sites available in the vector. We believe that these plasmids will be extremely useful in the gene expression studies in mycobacteria by offering the choices of promoters and reporters. Our work also paves the way to developing more such plasmids with other tags and promoters that may find use in mycobacterial biology.

SUBMITTER: Seniya SP

PROVIDER: S-EPMC7065818 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Construction of E. coli-Mycobacterium shuttle vectors with a variety of expression systems and polypeptide tags for gene expression in mycobacteria.

Seniya Surya Pratap SP Yadav Priya P Jain Vikas V

PloS one 20200311 3

Cloning and expression of a desired gene is indispensable in molecular biology studies. Expression vectors, in this regard, should offer much needed flexibility and choice of cloning strategies for both in vivo and in vitro protein expression experiments. Furthermore, availability of option to choose from various reporter tags allows one to be flexible during designing of an experiment in a more relevant manner. Thus, the need of a versatile expression system cannot be ignored. Although several ...[more]

PMID: 32160243

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Project description:BackgroundLactic acid bacteria (LAB) are a diverse group of Gram-positive bacteria, which are widely distributed in various diverse natural habitats. These are used in a variety of industrial food fermentations and carry numerous traits with utmost relevance to the food industry. Genetic engineering has emerged as an effective means to improve and enhance the potential of commercially important bacterial strains. However, the biosafety of recombinant systems is an important concern during the implementation of such technologies on an industrial scale. In order to overcome this issue, cloning and expression systems have been developed preferably from fully characterized and annotated LAB plasmids encoding genes with known functions.ResultsThe developed shuttle vector pPBT-GFP contains two theta-type replicons with a copy number of 4.4 and 2.8 in Pediococcus acidilactici MTCC 5101 and Lactobacillus brevis MTCC 1750, respectively. Antimicrobial "pediocin" produced by P. acidilactici MTCC 5101 and green fluorescent protein (GFP) of Aequorea victoria were successfully expressed as selectable markers. Heterologous bile salt hydrolase (BSH) from Lactobacillus fermentum NCDO 394 has been efficiently expressed in the host strains showing high specific activity of 126.12 ± 10.62 in P. acidilactici MTCC 5101 and 95.43 ± 4.26 in the case of L. brevis MTCC 1750, towards glycine-conjugated bile salts preferably as compared to taurine-conjugated salts.ConclusionThe present article details the development of a LAB/LAB shuttle expression vector pPBT-GFP, capable of replication in LAB hosts, P. acidilactici MTCC 5101, and L. brevis MTCC 1750. Pediocin and GFP have been used as selectable markers with the efficient production of heterologous extracellular bile salt hydrolase. Thus, the constructed vector pPBT-GFP, with its ability to replicate in multiple hosts, low copy number, and stability in host cells, may serve as an ideal tool for improving LAB strains of commercial value using genetic engineering.

Project description:Polyelectrolytes of opposite charge in aqueous solution can undergo a liquid-liquid phase separation known as complex coacervation. Complex coacervation of ampholytic proteins with oppositely charged polyelectrolytes is of increasing interest as it results in a protein rich phase that has potential applications in protein therapeutics, protein purification, and biocatalysis. However, many globular proteins do not phase separate when mixed with an oppositely charged polyelectrolyte, and those that do phase separate do so over narrow concentration, pH, and ionic strength ranges. The protein design factors that govern complex coacervation under varying conditions are still relatively unexplored. Recent work indicates that proteins with an intrinsically disordered region, a higher net charge, or a patch of charged residues are more likely to undergo a phase transition. Based on these design parameters, polyionic coacervation tags were designed and assessed for their ability to promote protein complex coacervation with oppositely charged polyelectrolytes. The phase behavior of a panel of engineered proteins was evaluated with the strong polycation poly(4-vinyl N-methyl pyridinium iodide). Proteins containing the ionic tags formed liquid coacervate droplets, while isotropically charged protein variants formed solid precipitates. The ionic tags also promoted phase separation at higher salt concentrations than an isotropic distribution of charge on the protein surface. The salt dependence of the protein complex coacervation could be predicted independently for tagged or isotropic variants by the ratio of negative-to-positive residues on the proteins and universally by calculating the distance between like charges. The addition of just a six residue polyionic tag generated a globular protein capable of liquid-liquid phase separation at physiological pH and ionic strength. This model system has provided the initial demonstration that short, ionic polypeptide sequences (6-18 amino acids) can drive the liquid-liquid phase separation of globular proteins.

Dataset Information

Construction of E. coli-Mycobacterium shuttle vectors with a variety of expression systems and polypeptide tags for gene expression in mycobacteria.

Publications

Construction of E. coli-Mycobacterium shuttle vectors with a variety of expression systems and polypeptide tags for gene expression in mycobacteria.

Similar Datasets

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