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

Designing an optimized strategy for extracellular expression of recombinant human TNF-α in Escherichia coli

Extracellular protein expression in Escherichia coli is an elegant solution that addresses the complex issue of protein misfolding while simultaneously simplifying downstream processing steps. Human TNF-α was chosen as the target protein for export since it is a therapeutically important cytokine. Different genomic knockouts were tested for the ability to sustain and enhance protein expression, and BW25113 Δ( elaA + cysW ) knockout was found to give a sustained and high level of expression. To improve secretion, various tags were tested, and the MBP tag at the N-terminal end was found to give maximum enhancement in the export of hTNF-α. Even the linker peptide was found to play a critical role in export, with the Ek linker giving the highest extracellular secretion, while the intein sequence completely blocked export. The co-expression of pSecAB, which is involved in protein transport to the periplasm, was also found to be helpful in enhancing extracellular protein titers. Interestingly, pelB performed poorly as compared to the native signal sequence of MBP, which gave better results. Culture conditions were optimized, and it was observed that growing cells in TB medium at a temperature of 25 °C, coupled with a pulse of concentrated nutrients at 24 h, led to a very high extracellular accumulation of ∼1.3 g/L of MBP-hTNF-α in shake flask culture. The protein was purified and tested using L929 cells for bioactivity. Thus, a combination of genomic and bioprocess strategies allowed us to obtain high levels of soluble and active extracellular expression of hTNF-α, making this a very attractive strategy for protein production.

An expression framework for production of recombinant methionine aminopeptidase in Escherichia coli: a case study of upstream process.

A case-study evaluation of a pBR322-derived expression system, previously applied to therapeutic peptides and insulin analogs, in combination with fed-batch cultivation for recombinant production of methionine aminopeptidase in E. coli, evaluating the performance of this system for methionine aminopeptidase as a stress-sensitive model protein under the tested conditions without a side-by-side comparison with alternative expression systems.

Efficient expression and purification of recombinant MUTYH protein in Escherichia coli

MUTYH is a DNA glycosylase involved in the excisional base repair (BER) process responsible for the recognition and removal of adenine improperly bonded to 8-oxoguanine. Pathogenic variants or impaired MUTYH function are associated with MUTYH-associated polyposis and an increased risk of colorectal cancer. Recombinant MUTYH production is necessary for biochemical and structural studies. In this study, we developed and optimized a protocol for the expression and purification of recombinant human MUTYH protein fused with the 6×His N-terminal label. Wild-type constructs were cloned into the pET-28c expression vector and expressed in Escherichia coli Rosetta (DE3), ArcticExpress(DE3), and SHuffle® T7 Express strains. Low-temperature induction and optimized IPTG concentrations improved protein solubility. Recombinant MUTYH was purified using Ni2⁺-affinity chromatography followed by affinity chromatography with heparin. SDS-PAGE and Western blot analyses confirmed a high level of expression and effective isolation of the recombinant protein, with a significant portion isolated in a soluble form. The optimized workflow provides a reproducible method for obtaining purified MUTYH, suitable for subsequent biochemical and structural studies. In the future, we plan to evaluate its enzymatic activity.

Recombinant expression of Zika virus NS1 protein in Escherichia coli: effects of fusion strategy and culture conditions.

Zika virus (ZIKV) nonstructural protein 1 (NS1) represents a valuable diagnostic target due to its early secretion during infection and reduced cross-reactivity with other flaviviruses. Optimization of NS1 expression conditions is essential for obtaining large-scale protein production. The use of bacterial expression systems as Escherichia coli offers a cost-effective platform. We developed two molecular cloning strategies to express full-length ZIKV NS1 (1053 bp) from Cuban strain 9661/2016 using pQE30/XL1-Blue and pET50b(+)/BL21(DE3) expression systems. The ns1 gene cloned via pGEM-T intermediate vector, and subcloned into expression vectors by using directional restriction-based strategy. The effects of culture medium and IPTG concentration on bacterial growth and protein accumulation was evaluated in order to optimizing the NS1 expression conditions. Both expression systems successfully produced recombinant ZIKV NS1. However, the BL21(DE3)/pET50b(+) system yielded soluble NS1, whereas the XL1-Blue/pQE30 system predominantly produced insoluble protein. Culture medium exerted a greater effect on protein expression than IPTG concentration, with LB medium and 0.1 mM IPTG providing the most favorable conditions for recombinant NS1 production. Notably, XL1-Blue demonstrated competency for protein expression. The pET50b(+)-ns1/BL21(DE3) system offers decisive advantages through soluble expression, eliminating refolding requirements for downstream purification process. This work establishes optimized protocols for ZIKV NS1 expression applicable to obtaining a recombinant protein for developing an ELISA system and broadly transferable to other flavivirus antigens.

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

A streamlined cloning platform for expressing a gene of interest either alone or fused to a monomeric fluorescent protein under the control of two promoters of choice is described, providing a fast and flexible cloning strategy for protein expression and subcellular localization analyses in mycobacteria.

CRISPR-Cpf1-Mediated T4 Phage Genome Editing for One-Step In Vivo Display of Heterologous Protein.