Aug 2026· Protein Expression and Purification· pp.
106996
· 0 citations· 49 references
Medicine
TL;DR
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.
Abstract
Incomplete removal of the initiator methionine is a frequent bottleneck in Escherichia coli-based production of recombinant proteins, causing heterogeneity and increased immunogenicity of biopharmaceuticals. Methionine aminopeptidase (MAP) is the key enzyme responsible for this post-translational modification, yet its endogenous activity is rapidly saturated under high-level expression conditions. Here, we report 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. The map gene from E. coli BL21(DE3) was cloned into the pF644 vector to generate pF1492. During fed-batch cultivation under the tested conditions, specific productivity reached 127.03 ± 8.66 mg·g-1 and volumetric productivity of total cell-associated MAP reached 2.71 ± 0.18 g·L-1 by the final hour of induction. MAP accumulated predominantly as insoluble inclusion bodies, which is a common outcome for recombinant protein expression in E. coli at high rates. This study reports upstream production and inclusion body formation only; functional recovery and enzymatic activity were not assessed. Acetate remained moderate (35-60 mM) and biomass was stable, indicating balanced metabolism. The present study evaluates 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.
Protein-glutamine glutaminase (PGG) is a promising enzyme for improving the functionality of plant proteins, but its industrial production is limited by low native yields and the need for proteolytic activation. Here, we developed a standardized modular Golden Gate-based secretion platform in
Bacillus subtilis
to screen promoter-signal peptide combinations for extracellular production of
Chryseobacterium proteolyticum
PGG (
Cp
PGG) within a common construct architecture. Reporter and enzyme-based screening identified Pgrac100-amyQ* as the configuration that consistently supported robust secretion and functional
Cp
PGG production in
B. subtilis
168, where the secreted proenzyme underwent host-mediated maturation. In 3-L batch bioreactor cultivation in rich medium, this construct reached 2.9 U/mL of supernatant (SN), whereas fed-batch cultivation in defined medium revealed extracellular proteolysis as a major limitation under high-cell-density conditions. To decouple secretion from activation, the construct was transferred to the protease-deficient strain KO7-S which enabled stable accumulation of non-processed
Cp
PGG. Subsequent controlled in vitro activation with a food-grade neutral protease yielded 12.8 ± 0.8 U/mL SN. Together, these results show that efficient PGG production in
B. subtilis
requires coordinated control of secretion, extracellular stability and proenzyme activation.
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Modular screening identified Pgrac100-amyQ* for CpPGG secretion in B. subtilis.
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Proteolysis limited high-cell-density fed-batch production in B. subtilis 168.
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In Bs KO7-S, secretion and activation were decoupled yielding 12.8 ± 0.8 U/mL SN.
Clàudia Lliso-Pascual, Sergi Abad, Marc Carnicer et al.· Applied Microbiology and Bio...· 0 citations
2'-Fucosyllactose (2'-FL), the most abundant human milk oligosaccharide (HMO), has attracted considerable interest for its prebiotic and immunomodulatory functions, with broad applications in infant nutrition. In this study, we report the development of a high-yield, genome-integrated 2'-FL-producing strain based on Escherichia coli MG1655 through systematic modular optimization. Starting from a single-copy BKHT strain (MGC06), we first optimized the copy number of the α-1,2-fucosyltransferase (α-1,2-FT) gene BKHT. Subsequently, the GDP-L-fucose supply was enhanced through coordinated genomic integration of the gene clusters cpsG-cpsB and gmd-fcl, while the multidrug efflux transporter gene mdfA was integrated to improve product export and strain robustness. BKHT copy number was then re-evaluated in the optimized background, with four copies yielding the highest production. The final engineered strain, harboring all genetic modifications stably integrated into the chromosome, produced 17.18 g/L 2'-FL in shake-flask culture. In fed-batch fermentation using a 5-L bioreactor, this strain achieved a titer of 154.12 g/L after 60 h, with a productivity of 2.57 g/L/h. Notably, throughout the entire fermentation process, no antibiotics or inducers were supplemented, underscoring the genetic stability and regulatory compliance of this plasmid-free system. To our knowledge, this represents the highest 2'-FL titer reported to date, positioning our engineered strain as a promising candidate for commercial 2'-FL production.
Rou-Lin Chen, Longhao Yang, Hao Wang et al.· Enzyme and Microbial Technol...· 0 citations
Carcinine is an imidazole dipeptide with potent antioxidant and antiglycation properties, although its chemical synthesis currently relies on severely environmentally harmful processes. In this work, a multi-enzyme cascade biotransformation system comprising 4′-phosphopantetheinyl transferase and non-ribosomal peptide synthetase was constructed. To overcome the limitations arising from stochastic spatial distribution and suboptimal mass transfer associated with independent enzymes, a fusion protein strategy was adopted. The two enzymes were fused via a flexible genetic linker within plasmid pET28a-SFP-L-Ebony, which enabled robust soluble expression in Escherichia coli. Concurrently, the endogenous peptidase genes (pepA, pepB, pepD, and pepN) were systematically knocked out using CRISPR/Cas9-mediated gene editing. This quadruple protease-deficient strain (designated SFP-L-Ebony-ΔpepABDN) effectively suppressed product degradation. Subsequent optimization revealed that optimal catalytic performance occurred at 25 °C and pH 7.0. The highest biotransformation efficiency was achieved using 15 g/L crude enzymes, in the presence of 2 mM ATP and 10 mM MgCl2. Through a fed-batch substrate feeding strategy in a 50 mL reaction system, the final carcinine titer reached 7.0 g/L after 48 h. This study, therefore, provides an efficient and sustainable technological pathway for the green biomanufacturing of carcinine as well as other high-value dipeptides.
This review summarizes recent advances in engineering key expression elements underlying heterologous protein production in K. phaffii, with particular emphasis on promoter architecture redesign, signal peptide replacement and sequence engineering, molecular chaperone co-expression, and quantitative regulation of the unfolded protein response.
Ru-Yue Han, Ruizheng Hu, An-Ran Liu et al.· Journal of Fungi· 1 citation
The present study evaluated the induction conditions (inducer type and concentration) for the expression of the multi-antigenic chimeric protein TgAGS/BsT from Toxoplasma gondii in Escherichia coli BL21 Star during batch submerged cultivation. With a molecular mass of about 18.6 kDa, the chimeric TgAGS/BsT protein has potential use in a diagnostic kit and as a vaccine candidate for toxoplasmosis. Batch cultures were performed at 37°C and induced by the addition of different concentrations of isopropyl β-D-1-thiogalactopyranoside (IPTG) (0.1, 1.0, and 2.0 mM) and lactose (1.0, 10.0, and 20.0 g/L) in different culture media (2xTY and Terrific Broth). The parameters used to analyze the cultures were cell growth, acetic acid formation, pH variation, plasmid stability, and protein expression evaluated by SDS-PAGE, together with spectrophotometric techniques. Higher concentrations of IPTG (2.0 mM) and lactose (10.0 and 20.0 g/L) promoted higher acetic acid accumulation and a continuous decrease in pH, indicating metabolic stress. The pH profile varied with the medium: 2xTY showed partial recovery due to acetate reabsorption, while TB exhibited a continuous decline. Plasmid stability remained high (>90%) across all evaluated conditions. The results demonstrate that the concentration and type of inducer, combined with medium composition, have a strong influence on the balance between cell growth, metabolism, and the efficiency of heterologous protein expression. Lactose induction provided a more gradual process and achieved higher volumetric protein concentrations in TB medium (0.039 g/L with 10.0 g/L lactose) and higher protein yield per biomass in 2xTY medium (0.0086 g/g with 10.0 g/L lactose), whereas increasing IPTG concentrations did not enhance protein accumulation.
S. Matias, José Daladiê Barreto da Costa, Andrew D Moura et al.· Protein Expression and Purif...· 0 citations
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.
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Proof-of-concept validation of the modular p2GUS expression system
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Tandem His₁₀–GST–His₁₀ architecture improves Ni-NTA retention and purification
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Multitagged p2GUS-derived mTFAM retained DNA-binding activity
Pedro Ferro-Gallego, Lourdes Domínguez-Gerpe· Applied Microbiology and Bio...· 0 citations
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