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Ribonuclease inhibitors (RIs) are an indispensable biotechnological tool for the detection and manipulation of RNA. Nowadays, due to the outbreak of COVID-19, highly sensitive detection of RNA has become more important than ever. Although the recombinant expression of RNase inhibitors is possible in E. coli, the robust expression is complicated by maintaining the redox potential and solubility by various expression tags. In the present paper we describe the expression of RI in baculovirus-infected High Five cells in large scale utilizing a modified transfer vector combining the beneficial properties of Profinity Exact Tag and pONE system. The recombinant RI is expressed at a high level in a fusion form, which is readily cleaved during on-column chromatography. A subsequent anion exchange chromatography was used as a polishing step to yield 12 mg native RI per liter of culture. RI expressed in insect cells shows higher thermal stability than the commercially available RI products (mainly produced in E. coli) based on temperature-dependent RNase inhibition studies. The endotoxin-free RI variant may also be applied in future therapeutics as a safe additive to increase mRNA stability in mRNA-based vaccines.

Crocins are important natural products predominantly obtained from the stigma of saffron, and that can be utilized as a medicinal compound, spice, and colorant with significant promise in the pharmaceutical, food, and cosmetic industries. Carotenoid cleavage dioxygenase 2 (CsCCD2) is a crucial limiting enzyme that has been reported to be responsible for the cleavage of zeaxanthin in the crocin biosynthetic pathway. However, the catalytic activity of CsCCD2 on β-carotene/lycopene remains elusive, and the soluble expression of CsCCD2 remains a big challenge. In this study, we reported the functional characteristics of CsCCD2, that can catalyze not only zeaxanthin cleavage but also β-carotene and lycopene cleavage. The molecular basis of the divergent functionality of CsCCD2 was elucidated using bioinformatic analysis and truncation studies. The protein expression optimization results demonstrated that the use of a maltose-binding protein (MBP) tag and the optimization of the induction conditions resulted in the production of more soluble protein. Correspondingly, the catalytic efficiency of soluble CsCCD2 was higher than that of the insoluble one, and the results further validated its functional verification. This study not only broadened the substrate profile of CsCCD2, but also achieved the soluble expression of CsCCD2. It provides a firm platform for CsCCD2 crystal structure resolution and facilitates the synthesis of crocetin and crocins.

Heterologous functional expression of the recombinant lipases is typically a bottleneck due to the expression in the insoluble fraction as inclusion bodies (IBs) which are in inactive form. Due to the importance of lipases in various industrial applications, many investigations have been conducted to discover suitable approaches to obtain functional lipase or increase the expressed yield in the soluble fraction. The utilization of the appropriate prokaryotic and eukaryotic expression systems, along with the suitable vectors, promoters, and tags, has been recognized as a practical approach. One of the most powerful strategies to produce bioactive lipases is using the molecular chaperones co-expressed along with the target protein’s genes into the expression host to produce the lipase in soluble fraction as a bioactive form. The refolding of expressed lipase from IBs (inactive) is another practical strategy which is usually carried out through chemical and physical methods. Based on recent investigations, the current review simultaneously highlights strategies to express the bioactive lipases and recover the bioactive lipases from the IBs in insoluble form.

The overexpression of proteins in Escherichia coli often results in the formation of inclusion bodies, which are biologically inactive, especially for proteins with exposed hydrophobic surfaces. Solubilization of inclusion bodies (IBs) and subsequent refolding is essential for obtaining correctly folded and active protein. However, protein refolding involves multiple steps—namely isolation, solubilization, and refolding—which is a labor-intensive process. In this study, we developed a strategy for soluble production and protein refolding. A fusion tag was applied to Burkholderia ambifaria lipase YCJ01, enabling abundant soluble expression in E. coli . Despite this, the soluble protein exhibited only partial enzymatic activity, suggesting an unfolded state of soluble lipase YCJ01. Lipase activity increased significantly after incubation with cosolvents, reaching 1003 U/mL, 754 U/mL, and 501 U/mL in 25% (v/w) glycerol, 15% (v/w) DMSO, and 4M trimethylamine N-oxide (TMAO) solutions, respectively. Correctly folded and highly active lipase YCJ01 with a natural N-terminus was obtained. Moreover, the cosolvent-induced refolding mechanism was elucidated through molecular dynamics simulations. Glycerol and DMSO were found to aggregate around hydrophobic regions of lipase, directly stabilizing structure by displacing water molecules and weakening water–protein hydrogen (H) bonds within the hydration shell. Conversely, TMAO molecules indirectly influenced the lipase structure by strengthening water–water H bonds. Key points • Cosolvents enhance lipase activity, with glycerol showing the highest improvement . • MD simulations show glycerol and DMSO directly interact with hydrophobic regions. • Glycerol and DMSO stabilize lipase directly, while TMAO enhances stability indirectly.

High soluble protein expression in heterologous hosts is crucial for various research and applications. Despite considerable research on the impact of codon usage on expression levels, the relationship between protein sequence and expression is often overlooked. In this study, a novel connection between protein expression and sequence is uncovered, leading to the development of SRAB (Strength of Relative Amino Acid Bias) based on AEI (Amino Acid Expression Index). The AEI served as an objective measure of this correlation, with higher AEI values enhancing soluble expression. Subsequently, the pre‐trained protein model MP‐TRANS (MindSpore Protein Transformer) is developed and fine‐tuned using transfer learning techniques to create 88 prediction models (MPB‐EXP) for predicting heterologous expression levels across 88 species. This approach achieved an average accuracy of 0.78, surpassing conventional machine learning methods. Additionally, a mutant generation model, MPB‐MUT, is devised and utilized to enhance expression levels in specific hosts. Experimental validation demonstrated that the top 3 mutants of xylanase (previously not expressed in Escherichia coli) successfully achieved high‐level soluble expression in E. coli. These findings highlight the efficacy of the developed model in predicting and optimizing gene expression based on protein sequences. Understanding the correlation between protein sequences and expression is essential for many research and applications. This study introduced a new mathematical model to confirm the association between protein sequence and expression. The pre‐trained protein model, MP‐TRANS, is developed and fine‐tuned using transfer learning techniques to create prediction models, as well as models for generating mutants to adjust soluble expression levels.

Background Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli . To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli . Results A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli . The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli : four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. Conclusions The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli .

Interleukin-37 is a cytokine with potent immunosuppressive properties that has been shown to have potential to treat autoimmune and chronic inflammatory diseases, as well as certain types of cancer. IL-37 is a 19 kDa protein which interacts with proteins in receptor-dependent and receptor-independent pathways. The expression of the IL-37 protein cloned into the pET-28a vector was optimized in Rosetta 2(DE3) after comparing its expression with Rosetta-gami 2(DE3) and Rosetta 2(DE3) pLysS, which was then used for the large-scale production of IL-37. IMAC purification of IL-37 yielded > 97% pure 0.9 mg/mL protein from auto-induced fermentation. The IC50 value of IL-37 was < 1 µM, which was similar to that of doxorubicin, and proliferation of > 80% of all cancer cells was inhibited by 100 µg/mL of IL-37 protein. IL-37 may be a promising theragnostic target for cancer due to its comparable IC50 value with that of doxorubicin.

Alginate lyases cleave the 1,4-glycosidic bond of alginate by eliminating sugar molecules from its bond. While earlier reported alginate lyases were primarily single catalytic domains, research on multi-module alginate lyases has been lfiguimited. This study identified VsAly7A, a multi-module alginate lyase present in Vibrio sp. QY108, comprising a “Pro-Asp-Thr(PDT)” fragment and two PL-7 catalytic domains (CD I and CD II). The “PDT” fragment enhances the soluble expression level and increases the thermostability and binding affinity to the substrate. Moreover, CD I exhibited greater catalytic efficiency than CD II. The incorporation of PDT-CD I resulted in an increase in the optimal temperature of VsAly7A, whereas CD II displayed a preference for polyG degradation. The multi-domain structure of VsAly7A provides a new idea for the rational design of alginate lyase, whilst the “PDT” fragment may serve as a fusion tag in the soluble expression of recombinant proteins.

Antimicrobial peptides (AMPs) are a kind of small molecular peptide that an organism produces to resist the invasion of foreign microorganisms. AMP BSN-37 is a bovine AMP that exhibits high antibacterial activity. In this paper, the optimized gene AMP BSN-37 was cloned into pCold-SUMO for fusion expression by recombinant DNA technology. The gene sequence of AMP BSN-37 was obtained by codons reverse translation, and the codons were optimized according to the codons preference of Escherichia coli (E. coli). The recombinant plasmid was constructed and identified by PCR, enzyme digestion and sequencing. Then the recombinant plasmid was transformed into BL21 E. coli to induce expression, and the IPTG concentration and time were optimized. The expressed soluble fusion protein SUMO-BSN-37 was purified by chromatography and then cleaved by SUMO proteases to release BSN-37. SDS-PAGE electrophoresis and Western blotting were used for identification. The recombinant plasmid pCold-SUMO-BSN-37 was obtained, and the fusion AMP BSN-37 was preliminarily expressed in BL21. After optimization, the optimal expression condition was 37 ℃ with 0.4 µM IPTG and 6 h incubation. Under optimal conditions, a large amount of fusion AMP BSN-37 was obtained by purification. Western blotting showed that the fusion peptide was successfully expressed and had good activity. The expressed BSN-37 showed antimicrobial activity similar to that of synthesized BSN-37. In this study, soluble expression products of AMP BSN-37 were obtained, and the problem regarding the limited source of AMP BSN-37 could be effectively solved, laying a foundation for further research on AMP BSN-37.

Ochratoxin A (OTA) is a globally distributed mycotoxin that poses serious threats to food safety and human health due to its nephrotoxic, hepatotoxic, and carcinogenic properties. Previous enzymatic detoxification strategies for OTA have been constrained by low degradation efficiency or poor soluble expression of highly active enzymes. In this study, a bacterial strain with strong OTA-degrading activity was isolated and identified as Acinetobacter kookii AK4, which degraded 95.44% of 1 μg/mL OTA within 6 h. The predominant OTA-degrading activity was derived from intracellular enzymes. Through genome mining and experimental validation, gene2102 was identified as encoding an amidohydrolase. The enzyme was designated AMH2102 and was heterologously expressed in Escherichia coli. Codon optimization combined with fusion of an N-terminal SUMO tag increased the soluble expression of AMH2102 by 14.81-fold, enabling complete (100%) OTA degradation within 3 min. Overall, this study achieved the identification of an efficient OTA-degrading strain and enzyme and explored strategies for improving enzyme expression, yielding effective outcomes that provide useful references for future studies on strain mining and enzyme engineering.