A simple multi-cloning strategy for gene expression and subcellular localization in Mycobacteria.

free cloning for high throughput gene cloning and expression

cerevisiae-based platform for rapid production and evaluation of Eukaryotic Nutrient transporters and transceptors for biochemical studies and crystallography

An integrated automated pipeline for cloning, transient expression, and purification augmenting the plant cell pack platform for biopharmaceutical screening.

Biopharmaceuticals, particularly monoclonal antibodies (mAbs), are a rapidly expanding class of therapeutics and have benefitted from high-throughput screening (HTS) strategies for protein engineering. Obtaining such complex biopharmaceuticals from plants might be advantageous because transient expression can yield grams of product within a week. For HTS, this system was scaled down as plant cell pack (PCP) technology to microtiter plate format and automated on a liquid-handling station, but is limited by manual cloning and protein purification. Therefore, we first generated a modular vector library comprising 15 pTRAc backbone constructs differing in regulatory gene expression and protein targeting elements, enabling rapid integration of any target gene via restriction-ligation cloning. Building on this, we developed an automated, seamless cloning workflow prior to transient expression and integrated a magnetic bead-based downstream purification process for single-chain fragment variable (scFv) mAbs. This automated workflow enables sequential cloning, expression, and purification of up to 375 protein variants simultaneously, each with 10 PCP replicates, within 9 days on a single platform. In contrast, manual processing allows only 15 variants with 10 replicates in the same period - a 25-fold increase in throughput, while costs decreased by 30%, from 79 € to 61 € per variant. Using a recombinant immunotoxin against acute myeloid leukemia based on the anti-CD64 H22 scFv as case study, a recovery of 63% and purity >95% were obtained, suitable for in vitro assays. Thus, integrating our cloning and purification workflows into the PCP platform establishes an automated pipeline that accelerates early biopharmaceutical development.

Development of an arabinose-inducible gene expression system for nontuberculous mycobacteria.

Nontuberculous mycobacteria (NTM) are emerging pathogens for which genetic tools remain limited. Here, we developed an arabinose-inducible gene expression system based on a modified pBAD24 vector adapted for mycobacterial hosts. The vector carries replication origins for mycobacteria and Escherichia coli, as well as selectable markers compatible with NTM. In Mycobacterium abscessus (Mycobacteroides abscessus), the system enabled dose-dependent induction of target gene expression by arabinose, as demonstrated by increased antibiotic resistance and quantitative RT-PCR analysis. Although basal expression was observed in the absence of arabinose, expression levels were tunable across arabinose concentrations. The system was also functional in Mycobacterium smegmatis (Mycolicibacterium smegmatis) and Mycobacterium bovis BCG, although the degree of basal expression varied among host species. These results establish a tunable inducible expression system for mycobacteria and provide a useful genetic tool for studies of NTM biology.

Proof-of-concept validation of p2GUS, a modular expression system for enhanced Ni-binding purification of functional recombinant proteins in Escherichia coli

Protein expression and purification remain critical steps in basic research, biotechnology, and industrial applications. Here, p2GUS, a modular Escherichia coli expression system generated through targeted modification of Precursor 1, a parental pBAD-derived vector, is described. The principal feature is a tandem His₁₀–GST–His₁₀ architecture designed to enhance interaction with Ni–NTA matrices while retaining compatibility with post-expression processing strategies. The GST module serves primarily as a spacer between the two polyhistidine tags while also contributing solubility-enhancing properties. Engineered PreScission and Enterokinase cleavage sites, enabling controlled post-expression processing and generation of alternative protein forms derived from the same multitagged recombinant protein. As a proof of concept, p2GUS was evaluated using a truncated mitochondrial transcription factor A [mTFAM(p.1_41del)]. Recombinant protein production, purification, protease-mediated processing, and DNA-binding activity were assessed using multitagged mTFAM(p.1_41del) as a model substrate. Purification outcomes were compared with those obtained using the parental construct Precursor 1, encoding a single-His 10 –mTFAM(p.1_41del). Under the experimental conditions examined, the p2GUS-derived construct, purified using a simple single-microcentrifuge tube Ni–NTA batch procedure, showed improved recovery and yielded a processed His 10 –EK–mTFAM(p.1_41del) of substantially higher purity than the corresponding His 10 –mTFAM(p.1_41del) produced by Precursor 1. The purified recombinant protein retained DNA-binding activity, supporting preservation of its functional properties following expression, purification and storage. These results support proof-of-concept validation of p2GUS as a modular system for recombinant protein production, purification, and controlled post-expression processing. Although validated here using a DNA-binding protein, broader applicability remains to be established through evaluation with additional recombinant proteins. • Proof-of-concept validation of the modular p2GUS expression system • Tandem His₁₀–GST–His₁₀ architecture improves Ni-NTA retention and purification • Multitagged p2GUS-derived mTFAM retained DNA-binding activity

Optimization of a Genome Editing System for Lactococci Based on a CRISPR-Associated Transposon