Recombinant Expression Research Articles

Epigenetic regulation of histone methyltransferase SUV39H1 on the expression of recombinant protein in CHO cells

Histone methylation–mediated epigenetic modification significantly influences gene transcription and expression regulation. This study examined the effects of histone 3 lysine 9 trimethylation (H3K9me3) methyltransferase SUV39H1 and its specific inhibitor chaetocin on recombinant protein expression in Chinese hamster ovary (CHO) cells. Results indicated that stable SUV39H1-knockdown CHO cells exhibited reduced H3K9me3 levels while showing increased expression of recombinant adalimumab (rADM) and human serum albumin (rHSA) by approximately 45% and 136%, respectively. Furthermore, treatment with 20 nM chaetocin, a SUV39H1-specific inhibitor, enhanced expression of enhanced green fluorescent protein (EGFP), rADM, and rHSA in CHO cells. These findings demonstrate that both stable SUV39H1 knockdown and pharmacological inhibition through chaetocin effectively reduce H3K9me3 modification levels in CHO cells while significantly boosting recombinant protein production. The results strongly suggest SUV39H1’s critical regulatory role in recombinant protein expression within CHO cell systems. This research establishes a methodological foundation for developing engineered cell lines and optimizing high-efficiency CHO expression systems through cell engineering approaches.Key points•SUV39H1 knockdown boosted recombinant protein expression and decreased H3 K9 me3 levels.•Treatment with the SUV39H1-specific inhibitor chaetocin (20 nM) enhanced recombinant protein expression.•It provides a basis for developing efficient epigenetically regulated CHO expression systems.

Protein Ligases: Nature’s Gift for Protein/Peptide Synthesis

Proteins are structurally and functionally diverse biomacromolecules that serve a variety of essential activities to ensure complex biological homeostasis. The desire to elucidate and enhance these biological functions has been at the forefront of research for many decades. However, generating active proteins via recombinant expression or through chemical total synthesis each has limitations in terms of yield and functionality. Nature has provided a solution to this problem through evolving protein ligases that catalyze the formation of amide bonds between peptides/proteins, which can be exploited by protein engineers to develop robust functional proteins. Here, we summarize the biochemical mechanisms and applications of multiple cysteine-based protein ligases, especially focusing on how they have been utilized for protein therapeutics and engineering, as well as how they inspired chemists to develop efficient methodologies for protein synthesis (e.g., native chemical ligation).

Overexpression of Tgm2 in Chinese Hamster Ovary Cells Enhances Recombinant Monoclonal Antibody Expression and Promotes Cell Proliferation through Reduction of Apoptosis.

Chinese hamster ovary (CHO) cells are the preferred host system for producing protein-based (antibody) therapeutics. However, recombinant CHO cells undergo substantial apoptosis during prolonged cultivation, impairing cell growth and ultimately compromising product yield and quality. Transglutaminase 2 (Tgm2), which mediates post-translational modifications of substrate proteins, regulates critical biological processes including cellular differentiation, apoptosis, cell cycle progression, and extracellular matrix assembly. In this study, we examined the effects of Tgm2 overexpression and knockdown on CHO cell growth and recombinant antibody production. Stable Tgm2 overexpression enhanced CHO cell proliferation while reducing apoptotic rates, resulting in significantly increased recombinant adalimumab expression (2.09 ± 0.08-fold) and specific productivity (1.88 ± 0.08-fold) compared to controls. In contrast, Tgm2 knockdown promoted apoptosis and induced cell cycle arrest. Mechanistically, elevated Tgm2 upregulated antiapoptotic genes (Bcl-2, Bcl-xL, and Mcl-1) while suppressing caspase-3 activity and BAX expression. These effects were associated with PI3K/AKT/mTOR pathway activation. Our findings demonstrate that Tgm2 overexpression enhances proliferation, bolsters antiapoptotic capacity, and improves monoclonal antibody production efficiency in CHO cells, establishing it as a viable strategy for increasing recombinant protein yields.

M6A modification profiles of the CHO cells with differential recombinant protein expression using MeRIP-seq/RNA-seq.

m6A modification profiles of the CHO cells with differential recombinant protein expression using MeRIP-seq/RNA-seq.

Improved recombinant protein expression using the 5'-untranslated region in Chinese hamster ovary cells.

Improved recombinant protein expression using the 5'-untranslated region in Chinese hamster ovary cells.

Recombinant extracellular expression of β-glucosidase in Bacillus subtilis and its potential secretion mechanism.

Recombinant extracellular expression of β-glucosidase in Bacillus subtilis and its potential secretion mechanism.

Magnesium ions enhance biogenic amine degradation by Pichia kudriavzevii MZ5: Insights from transcriptomics and novel recombinant enzyme expression.

Magnesium ions enhance biogenic amine degradation by Pichia kudriavzevii MZ5: Insights from transcriptomics and novel recombinant enzyme expression.

Neonatal Fc receptor participates in endocytosis of Fc fusion protein in vivo and in vitro.

Neonatal Fc receptor participates in endocytosis of Fc fusion protein in vivo and in vitro.

Heterologous expression of maize-derived antimicrobial peptide ZmES4 in E. coli for potential plant pathogen control

ZmES4 is a plant-derived antimicrobial peptide (AMP) from maize that shows promise as a biocontrol agent against plant pathogenic organisms. In the context of growing challenges in sustainable agriculture, AMPs like ZmES4 represent innovative alternatives to chemical pesticides. This study focuses on the structural characterization and heterologous expression of the ZmES4 peptide in Escherichia coli (E. coli). The gene encoding ZmES4 was obtained from the maize female gametophyte (NCBI Reference Sequence: NM_001112150.3) and cloned into the pET24d(+) expression vector using NcoI and XhoI restriction sites. Transformation into E. coli BL21 (DE3) cells enabled recombinant expression upon induction with isopropyl β-D-1-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Bradford assays confirmed the expression of ORF-ZmES4, with protein concentrations ranging from 14.647 to 63.606 mg/mL. The successful expression of ZmES4 in E. coli highlights its potential application as a recombinant AMP for future plant disease management strategies.

Production of recombinant coiled coil silk proteins for materials synthesis.

Production of recombinant coiled coil silk proteins for materials synthesis.

High level food-grade expression of maltogenic amylase in Bacillus subtilis through genomic integration and comA overexpression.

High level food-grade expression of maltogenic amylase in Bacillus subtilis through genomic integration and comA overexpression.

Functional analysis of retinal-binding protein in the visual system of the nocturnal marine shellfish Pacific abalone (Haliotis discus hannai).

Functional analysis of retinal-binding protein in the visual system of the nocturnal marine shellfish Pacific abalone (Haliotis discus hannai).

StxB fusion strategy for immunogenic enhancement: Recombinant expression of AcAMP antimicrobial peptide.

StxB fusion strategy for immunogenic enhancement: Recombinant expression of AcAMP antimicrobial peptide.

The response surface method enables efficient optimization of induction parameters for the production of bioactive peptides in fed-batch bioreactors using Escherichia coli.

The production of recombinant peptides is critical in biotechnology and medicine for treating a variety of diseases. Thus, there is an urgent need for the development of quick, scalable, and cost-effective recombinant protein expression strategies. This study optimizes induction conditions for an insulin precursor, an analog GLP-1 precursor, and a peptide for COVID-19 therapy expression in E. coli using the response surface method. Factors such as pH, temperature, induction time, isopropyl-β-D-thiogalactopyranoside concentration, and optical density significantly influence peptide productivity. Experimental validation supports the effectiveness of these models in predicting peptide yields under optimal conditions. The optimal induction conditions were determined as follows: temperature at 37°C, pH of the medium 7.0-8.0, induction at the early logarithmic phase of growth, isopropyl-β-D-thiogalactopyranoside concentration of 0.05mM, and induction time of 6h. After model validation, the productivity of each peptide producer exceeded 3g/L. The optimal conditions achieved peptide titers significantly higher than those previously reported, suggesting that this technique is a versatile cultivation technology for the efficient production of different recombinant peptides. In conclusion, our research enhances the understanding of how tailored cultivation conditions can optimize recombinant peptide production efficiency.

Construction and characterization of single-framework fully synthetic nanobody libraries

This study is designed to address the development, synthesis, and screening of non-animal-derived nanoantibody libraries. Furthermore, it seeks to elucidate the impact of framework region selection and complementarity-determining region (CDR) design on the characteristics of synthesized nanoantibody libraries. These investigations aim to establish a robust theoretical and technical foundation for enhancing the efficacy, diversity, and practical applicability of synthetic nanoantibody libraries. In this study, a new framework (IGHV3S65*01-IGHJ4*01) was identified based on the high-throughput sequencing results of natural nanobodies, and degenerate primers were designed based on the frequency of amino acids at each position in the complementarity-determining region (CDR) region to synthesize the coding fragments of nanobodies by overlap PCR. After 40 times of electro-transformation, a single-frame synthesized nanobody library (SS-Library) containing 6×109 clones was obtained, and the titer of the library was demonstrated to be 1013 PFU/mL after rescue by the helper phage M13K07. Random 48 single colonies were picked for PCR, which revealed an insertion rate of 95.8%. Sanger sequencing results showed that 38 clones had complete sequences, none of which showed cysteines or stop codons, and no identical sequences appeared, suggesting that the library had higher diversity. The library was screened and validated with three antigens, including bovine serum albumin (BSA), acetylcholinesterase (AchE), and immunoglobulin G (IgG). Finally, 2 nanobodies against BSA, 10 against AchE, and 15 against IgG were obtained. One positive clone of each antigen was singled out for recombinant expression, and the results showed that all the three nanobodies were expressed in a soluble form. The binding activity of recombinantly expressed nanobodies was evaluated using indirect enzyme-linked immunosorbent assay (ELISA) and bio-layer interferometry (BLI). The results demonstrated that the anti-AChE and anti-IgG nanobodies exhibited specific binding to their respective antigens, with affinity constants (KD) of 294 nmol/L and 250 nmol/L, respectively. The nanobody synthetic library preparation method proposed in this study is simple and easy to use with low preference, and it is expected to be a universal nanobody discovery platform for the preparation and development of lead specific nanobodies.

Abstract 6742: High-affinity human IgG1 antibody against GUCY2C for ADC development: Identification, characterization, and efficacy

Abstract Colorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer mortality worldwide. Despite the early disease screening techniques could reduce the number of deaths, the treatments for metastatic CRC still remains limited. GUCY2C, a receptor predominantly found in the intestinal epithelium, is a tumor-associated antigen that is overexpressed in gastrointestinal malignancies, particularly colorectal cancer (CRC). GUCY2C offers a super clean therapeutic target, due to restricted expression in normal tissues and high expression in CRC cells, with significantly reduced the on-target-off-tumor toxicities. A high-affinity, high-specificity human IgG1 monoclonal antibody against GUCY2C was obtained from a 100 billion-capacity human Fab phage display library. This antibody, after recombinant expression in CHO cells, was extensively validated both in vitro and in vivo for its suitability in antibody-drug conjugate (ADC) development. The recombinant antibody demonstrated high affinity for its specific antigen, as confirmed by Bio-Layer Interferometry (BLI) and Enzyme-Linked Immunosorbent Assay (ELISA) methods. Flow cytometry further confirmed that the antibody had picomolar (pM) affinity for gastric and colorectal cancer cell lines expressing high levels of GUCY2C. Cellular internalization experiments indicated that the antibody possesses excellent endocytic capabilities, making it particularly suitable for ADC development. The antibody was then conjugated with a linker-payload complex containing a topoisomerase I inhibitor and an antimetabolite moiety, and subjected to a series of in vitro and in vivo evaluations. The ADC achieved in vitro proliferation inhibition in the picomolar range of IC50. Several in vivo CDX model experiments also demonstrated significant tumor suppression efficacy. Moreover, acute toxicity evaluation of the GUCY2C-ADC in diverse preclinical models indicated favorable safety profiles and tolerability. Overall, these compelling preclinical data supports the promising therapeutic potential of this ADC in targeted treatment of CRC. Citation Format: Yunxia Zheng, Xiafen Wu, Junxiang Jia, Huihui Guo, Xiangfei Kong, Gengxiang Zhao, Yuanyuan Huang, Hangbo Ye, Xiaolei Liu, Yi Luo, Xiaoqiang Xie, Xia Zhou, Lu Bai, Wenjun Li, Binbin Chen, Qingliang Yang, Robert Yongxin Zhao. High-affinity human IgG1 antibody against GUCY2C for ADC development: Identification, characterization, and efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6742.

Creation of a highly stable direct electron transfer-type enzyme sensor by combining a hyperthermophilic dehydrogenase and natural electron mediator

This study aimed to address the stability limitations of third-generation biosensors using enzymes from mesophilic organisms, by engineering a stable direct electron transfer (DET)-type dehydrogenase capable of transferring electrons extracted from the substrate to the electrode. A fusion protein combining the mediated electron transfer (MET)-type aldose sugar dehydrogenase from the hyperthermophile Pyrobaculum aerophilum (PaeASD), which cannot transfer electrons generated by enzymatic reactions to the electrode without a mediator, and the natural electron transfer protein cytochrome b562 (cyt b562) was developed to investigate its potential for the DET reaction. A recombinant protein expression system was established in Escherichia coli to produce the PaeASD-cyt b562 fusion protein, which was purified from the soluble fraction of the host cells. Intramolecular electron transfer from pyrroloquinoline quinone (PQQ) to the heme group within the PaeASD-cyt b562 fusion protein was investigated using UV–Vis absorption spectroscopy. Upon the addition of glucose, an increase in absorption corresponding to reduced heme molecules was observed, indicating electron transfer from glucose to the heme group in the cyt b562 component via PQQ in the PaeASD component. The DET capability of the fusion protein was evaluated using cyclic voltammetry with screen-printed carbon electrodes. A glucose concentration-dependent increase in current response confirmed DET activity. Notably, the fusion protein retained over 80% of its initial current response even after 2 months of storage at 4 °C. The novel robust PaeASD-cyt b562 fusion protein demonstrated efficient DET capability, highlighting its high potential for application in the development of third-generation biosensors.

Design and application of expression constructs for FMDV serotype O structural proteins.

Design and validate flexible constructs for recombinant expression of FMDV serotype O structural proteins of the circulating topotypes using newly designed degenerate primers. The designed degenerate primers targeting diverse topotypes enabled the successful amplification of VP0, VP1, and VP3 genes. Integration of the essential transcriptional and translational regulatory elements including T7 promoter, leader g10 sequence, and T7 terminator, as well as ribosome binding site (RBS), start and stop codons, respectively via overlap extension PCR empowered efficient expression of these proteins in E. coli. Cloned constructs expressed the target proteins of expected molecular weights: VP0 (34kDa), VP1 (24kDa), and VP3 (22kDa). SDS-PAGE and Western blotting confirmed high protein yield and purity. This platform demonstrated adaptability for diagnostic and vaccine development applications. The workflow offers a robust tool for producing FMDV structural proteins concerning the circulating strains attempting to improve control measures including diagnosis and vaccinations.

Abstract 4494: Structure, dynamics and therapeutic potential of fatty acid synthase

Abstract De novo lipid synthesis is facilitated by the multifunctional enzyme fatty acid synthase (FASN), a hallmark of cancer associated with chemotherapeutic resistance and poor prognosis. Human FASN (hFASN) is a dynamic protein that undergoes significant conformational changes during catalysis, posing challenges for structural characterization. To date, no structural information is available for full-length hFASN, hindering our understanding of its catalytic mechanism and complicating rational drug design.Here, we report the structure of hFASN resolved using electron cryo-microscopy (cryoEM). The modifying region of hFASN was determined at near-atomic resolution using a recombinant expression system bound to the first-in-class inhibitor Denifanstat, enabling structure-based small molecule discovery. Additionally, we solved the full-length structure of endogenous hFASN from MCF7 cells, revealing an interaction with the small heat shock protein HSP27. In some 2D classes of hFASN from cryoEM data, we found partial unfolding of the modifying region, evidence of potential protein degradation. However, HSP27 knockdown in breast cancer cell lines did not alter overall hFASN levels, indicating that this interaction may be transient or involve additional partners.Mass spectrometry-based post-translational modification analysis further identified dihydroxylation of proline, arginine, and tyrosine residues in hFASN from MCF7 cells, likely reflecting oxidative stress. These modifications may play a role in protein stabilization or regulation under stress conditions, highlighting a potential mechanism for hFASN activity modulation. Together, these findings provide critical structural and regulatory insights, paving the way for targeted therapeutic development. Citation Format: Naimul Hasan, Alexander Keszei, Matthew Waas, Thomas Kislinger, Razq Hakem, Mohammad Mazhab-Jafari. Structure, dynamics and therapeutic potential of fatty acid synthase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4494.

Epileptic encephalopathy in a young Bengal cat caused by CAD deficiency

Developmental and epileptic encephalopathy type 50 (DEE50) in humans is a severe early-onset neurometabolic disorder caused by biallelic loss-of-function variants in the CAD gene encoding a key multi-enzymatic protein for de novo pyrimidine nucleotide synthesis. Untreated, the condition is often fatal, but patients respond to uridine supplementation, which fuels nucleotide synthesis through CAD-independent salvage pathways. Here, we report a novel variant in the feline CAD gene in a 4-month-old Bengal kitten with intractable seizures and abnormal behavior. The variant, XP_011279586.1:p.(Ser2015Asn), was predicted to affect the oligomerization of the C-terminal aspartate transcarbamylase (ATCase) domain of CAD. Genotyping of 110 unaffected Bengal cats revealed four additional carriers of the mutant allele, confirming its presence in the breed. In a CAD-knockout human cell line dependent on uridine, the recombinant expression of human wildtype CAD, but not of the Asn2015 mutant, restored cell growth without uridine, demonstrating that the p.Ser2015Asn variant disrupts CAD function and is pathogenic. This study facilitates genetic testing of carriers and affected cats and suggests that uridine supplementation could be a potential treatment. Furthermore, CAD-deficient Bengal cats might serve as a valuable spontaneous large animal model to further investigate the pathogenic mechanisms of this rare epileptic encephalopathy in humans.