Engineered polymer nanoparticles as artificial chaperones facilitating the selective refolding of denatured enzymes.

Molecular chaperones assist in protein refolding by selectively binding to proteins in their nonnative states. Despite progress in creating artificial chaperones, these designs often have a limited range of substrates they can work with. In this paper, we present molecularly imprinted flexible polymer nanoparticles (nanoMIPs) designed as customizable biomimetic chaperones. We used model proteins such as cytochrome c, laccase, and lipase to screen polymeric monomers and identify the most effective formulations, offering tunable charge and hydrophobic properties. Utilizing a dispersed phase imprinting approach, we employed magnetic beads modified with destabilized whole-protein as solid-phase templates. This process involves medium exchange facilitated by magnetic pulldowns, resulting in the synthesis of nanoMIPs featuring imprinted sites that effectively mimic chaperone cavities. These nanoMIPs were able to selectively refold denatured enzymes, achieving up to 86.7% recovery of their activity, significantly outperforming control samples. Mechanistic studies confirmed that nanoMIPs preferentially bind denatured rather than native enzymes, mimicking natural chaperone interactions. Multifaceted analyses support the functionality of nanoMIPs, which emulate the protective roles of chaperones by selectively engaging with denatured proteins to inhibit aggregation and facilitate refolding. This approach shows promise for widespread use in protein recovery within biocatalysis and biomedicine.

Characterization of structural and functional properties of soybean 11S globulin during renaturation after denaturation induced by changes in pH.

BACKGROUND: This study explored the denaturation of 11S globulin, a protein known for its diverse functional properties in soy protein applications, at pH 3.0 and pH 10.0, followed by a gradual return to pH 7.0 to facilitate renaturation. It investigated the structural and functional changes during renaturation induced by a change in pH, revealing the stabilization mechanism of 11S globulin. RESULTS: The findings revealed that during pH adjustment to neutral, the denatured soybean 11S globulin - resulting from alkaline (pH 10.0) or acidic (pH 3.0) treatments - experienced a refolding of its extended tertiary structure to varying extents. The particle size and the proportions of α-helix and ß-sheet in the secondary structure aligned progressively with those of the natural-state protein. However, for the alkali-denatured 11S, the ß-sheet content decreased upon adjustment to neutral, whereas an increase was observed for the acid-denatured 11S. In terms of functional properties, after alkaline denaturation, the foaming capacity (FC) and emulsifying activity index (EAI) of 11S increased by 1.4 and 1.2 times, respectively, in comparison with its native state. The solubility, foamability, and emulsifiability of the alkali-denatured 11S gradually diminished during renaturation but remained superior to those of the native state. Conversely, these properties showed an initial decline, followed by an increase during renaturation triggered by pH neutralization. CONCLUSIONS: This research contributes to the enhancement of protein functionality, offering a theoretical foundation for the development of functional soy protein products and expanding their potential applications. © 2024 Society of Chemical Industry.

Prokaryotic expression and functional analysis of molecular chaperone Acr2 protein of Mycobacterium tuberculosis / 中国生物制品学杂志

@#Objective To express the molecular chaperone Acr2 protein of Mycobacterium tuberculosis(Mtb)in E.coli and analyze the function. Methods The recombinant plasmid pET-28a-Acr2 was transformed into competent E. coli BL21(DE3),and induced by IPTG. The expressed His-Acr2 protein was purified by Ni-NTA chromatography and SuperdexTM200 10/300 GL gel filtration chromatography to obtain Acr2 protein. The Acr2 protein was refolded by spontaneous refolding and reassembly after thermal denaturation(100 ℃ for 15 min)and chemical denaturation(8 mol/L urea,37 ℃ for 4 h).The secondary structure of Acr2 protein before and after denaturation-renaturation was detected by circular dichroism spectroscopy and non-denaturing SDS-PAGE,and the molecular chaperone function of Acr2 protein in vitro was detected by substrate binding assay. Results The purified Acr2 protein had the relative molecular mass of about 232 000,the purity of over 90%,and the concentration of about 2 mg/mL,which recovered its natural secondary structure after denaturationrenaturation,and formed stable complexes with the denatured malate dehydrogenase(MDH)at 48 ℃. Conclusion The Acr2protein can restore its natural molecular conformation with molecular chaperone activity in vitro after denaturation-renaturation treatment,providing a new strategy for the preparation of Mtb protein antigen with natural activity.

Optimization of expression and purification conditions of recombinant Hq001 protein in salivary glands of Haemaphysalis qinghaiensis / 中国生物制品学杂志

@#Objective To optimize the expression conditions(expression and induction conditions)and purification methods(non-denaturing and denaturing purification)of recombinant Hq001 protein in salivary glands of Haemaphysalis qinghaiensis.Methods The recombinant plasmid pET-30a-Hq001 was transformed into competent cells E.coil BL21(DE3),E.coil Rosetta(DE3)and E.coil ArcticExpress(DE3)pRARE2 respectively for the selection of an optimal expression strain.The final concentration of IPTG(0,0.5,1.0 mmol/L),induction temperature(20,25 ℃)and induction time(0,2,4,6,8 h)were optimized.The recombinant bacteria expressed under the ideal induction condition were homogenized by French press and the target protein was purified by passing through a Ni-NTA affinity chromatography column under either native(denaturationrenaturation-column chromatography)or denatured conditions(denaturation-column chromatography-renaturation).The purified products were analyzed by 12% SDS-PAGE.Results E.coil BL21(DE3)was proved to be the most suitable strain for the expression of recombinant Hq001 protein.The optimum induction condition was induction with 0.5 mmol/L IPTG for 4 h at 25 ℃.The target protein with a relative molecular mass of approximately 18 800 was obtained by non-denaturing purification method,and the size was consistent with the expectation.Conclusion The recombinant protein rHq001 in salivary glands of Haemaphysalis qinghaiensis can be obtained by the optimized expression conditions and purification methods.

The return of nature? Negotiating the 'renaturation' of the Isar as an envirotechnical landscape.

How can we trace differing normative values, and especially in alternative imaginaries of environmentally sustainable futures? To address this issue, this article extends the sociotechnical imaginaries framework by providing conceptual tools to understand the underlying rationale of alternative environmental imaginaries-through an envirotechnical analysis. I analyse an urban river restoration project called the Isar-Plan in Munich, Germany, where the notion of 'renaturation' was at the centre a controversy over designs for the project. By positing the river as an envirotechnical landscape, the normative dimensions of nature, science and technology within environmental transformations can be constructively integrated within co-productionist analyses in science and technology studies. The article shows how existing societal values are shaped by prior systems and regimes, constructing local imaginaries of desirable environmental futures. Envirotechnical analyses also increase our ability to identify differing normative values, and could thus be further applied in cases where the normative assumptions behind opaque notions otherwise would be left underexplored.

Sulfated Polysaccharides as a Fighter with Protein Non-Physiological Aggregation: The Role of Polysaccharide Flexibility and Charge Density.

Proteins can lose native functionality due to non-physiological aggregation. In this work, we have shown the power of sulfated polysaccharides as a natural assistant to restore damaged protein structures. Protein aggregates enriched by cross-ß structures are a characteristic of amyloid fibrils related to different health disorders. Our recent studies demonstrated that model fibrils of hen egg white lysozyme (HEWL) can be disaggregated and renatured by some negatively charged polysaccharides. In the current work, using the same model protein system and FTIR spectroscopy, we studied the role of conformation and charge distribution along the polysaccharide chain in the protein secondary structure conversion. The effects of three carrageenans (κ, ι, and λ) possessing from one to three sulfate groups per disaccharide unit were shown to be different. κ-Carrageenan was able to fully eliminate cross-ß structures and complete the renaturation process. ι-Carrageenan only initiated the formation of native-like ß-structures in HEWL, retaining most of the cross-ß structures. In contrast, λ-carrageenan even increased the content of amyloid cross-ß structures. Furthermore, κ-carrageenan in rigid helical conformation loses its capability to restore protein native structures, largely increasing the amount of amyloid cross-ß structures. Our findings create a platform for the design of novel natural chaperons to counteract protein unfolding.

The effect of thioredoxin and prochymosin coexpression on the refolding of recombinant alpaca chymosin.

The milk-clotting enzyme chymosin is a member of the group of aspartate proteinases. Chymosin is the main component of rennet traditionally obtained from the stomachs of dairy calves and widely used to coagulate milk in the production of various types of cheese. Another source of chymosin, which does not require the killing of animals, is based on recombinant DNA technology. Recombinant alpaca chymosin has a number of valuable technological properties that make it attractive for use in cheese-making as an alternative to recombinant bovine chymosin. The purpose of this work is to study the effect of coexpression of thioredoxin and prochymosin on the refolding of the recombinant zymogen and the activity of alpaca chymosin. To achieve this goal, on the basis of the pET32a plasmid, an expression vector was constructed containing the thioredoxin A gene fused to the N-terminal sequence of the marker enzyme zymogen, alpaca prochymosin. Using the constructed vector, pET-TrxProChn, a strain-producer of the recombinant chimeric protein thioredoxin-prochymosin was obtained. The choice of prochymosin as a model protein is due to the ability of autocatalytic activation of this zymogen, in which the pro-fragment is removed, together with the thioredoxin sequence attached to it, with the formation of active chymosin. It is shown that Escherichia coli strain BL21 transformed with the pET-TrxProChn plasmid provides an efficient synthesis of the thioredoxin-prochymosin chimeric molecule. However, the chimeric protein accumulates in inclusion bodies in an insoluble form. Therefore, a renaturation procedure was used to obtain the active target enzyme. Fusion of thioredoxin capable of disulfide-reductase activity to the N-terminal sequence of prochymosin provides optimal conditions for zymogen refolding and increases the yield of recombinant alpaca chymosin immediately after activation and during long-term storage by 13 and 15 %, respectively. The inclusion of thioredoxin in the composition of the chimeric protein, apparently, contributes to the process of correct reduction of disulfide bonds in the prochymosin molecule, which is reflected in the dynamics of the increase in the milk-clotting activity of alpaca chymosin during long-term storage.

Enhanced Inhibition of Amyloid Formation by Heat Shock Protein 90 Immobilized on Nanoparticles.

As the population ages, an epidemic of neurodegenerative diseases with devastating social consequences is looming. To address the pathologies leading to amyloid-related dementia, novel therapeutic strategies must be developed for the treatment or prevention of neural protein-folding disorders. Nanotechnology will be crucial to this scenario, especially in the design of nanoscale systems carrying therapeutic compounds that can navigate the nervous system and identify amyloid to treat it in situ. In this line, we have recently designed a highly simplified and versatile nanorobot consisting of a protein coating based on the heat shock protein 90 (Hsp90) chaperone that not only propels nanoparticles using ATP but also endows them with the extraordinary ability to fold and restore the activity of heat-denatured proteins. Here, we assess the effectiveness of these nanosystems in inhibiting/reducing the aggregation of amyloidogenic proteins. Using Raman spectroscopy, we qualitatively and quantitatively analyze amyloid by identifying and semi-quantifying the Amide I band. Our findings indicate that the coupling of Hsp90 to nanoparticles results in a more potent inhibition of amyloid formation when compared to the soluble protein. We propose that this enhanced performance may be attributed to enhanced release-capture cycles of amyloid precursor oligomers by Hsp90 molecules nearby on the nanosurface. Intelligent biocompatible coatings, like the one described here, that enhance the diffusivity and self-propulsion of nanoparticles while enabling them to carry out critical functions such as environmental scanning, identification, and amyloid prevention, present an exceptional opportunity for the development of advanced nanodevices in biomedical applications. This approach, which combined active biomolecules with synthetic materials, is poised to reveal remarkable prospects in the field of nanomedicine and biotechnology.

Hydrated Ionic Liquids: Perspective for Bioscience.

Hydrated ionic liquid (IL) is a simple mixture of IL and water. Unique aqueous electrolyte solution can be designed by mixing IL with limited amount of water. In most hydrated ILs, there are no free water and all are strongly interacted with ions. The properties of hydrated ILs, such as polarity, viscosity, ion mobility, and hydrogen bonding ability, can therefore be controlled simply by water content. This mixture is expected to provide similar environment to that of living cell, and is desired to be effective solvents for biomolecules. In this account, we would like to survey the basic properties, recent results, and future aspects of the hydrated ILs.

Carboxylated chitosan improved the stability of phycocyanin under acidified conditions.

Phycocyanin, a natural blue colorant, derived from Spirulina platensis, is now widely used in the food industry. However, its main drawbacks are loss of color and denature of structure in an acidic environment. In this study, carboxylated chitosan (0.1 %-1 % w/v) was chosen as an additive in acid-denatured phycocyanin for preserving phycocyanin's blue color and natural structure. Zeta-potential and particle size revealed that the carboxylated chitosan with high negative charge adsorbed on phycocyanin and provided stronger electrostatic repulsion to overcome the protein aggregation. Ultraviolet-visible absorption spectrum and fluorescence spectroscopy showed that the carboxylated chitosan recovered the microenvironment of tetrapyrrole chromophores and ß-subunits, which led the secondary structure changed and the trimers depolymerized into the monomers changed by the acidic environment. Furthermore, Fourier transform infrared spectroscopy revealed highly negatively charged carboxylated chitosan with the groups (NH2, COOH and OH) could restored the microenvironment of tetrapyrrole chromophores and ß-subunits of phycocyanin, and interact with phycocyanin through hydrogen bonding, NH bonding, ionic bonding and van der Waals, which led to a change in secondary structure and depolymerization of trimers into monomers. Our study demonstrated the carboxylated chitosan played a beneficial role in recovering the structure of acid-denatured phycocyanin and its blue color.

Offsetting environmental impacts beyond climate change: the Circular Ecosystem Compensation approach.

Since the Paris Agreement entered into force, climate neutrality and associated compensation schemes are even more on the agenda of politics and companies. Challenges of existing offsetting schemes include the rather theoretical saving scenario and the limited scope of considered impacts. To address some of these limitations, this paper proposes the Circular Ecosystem Compensation (CEC) approach based on monetization of LCA results and Ecosystem Valuation. CEC consists of six steps: i) carrying out a life cycle assessment, ii) reducing the environmental impacts, iii) determining environmental costs applying monetization methods, iv) deriving the environmental value based on restoration costs methods, v) implementing the ecological restoration of ecosystems and vi) monitoring of the renaturation measures. Thus, CEC allows to offset a broad set of environmental impacts beyond climate change (e.g., acidification, eutrophication, land use, water use) in a real ecosystem by renaturation of degraded ecosystems. Environmental burdens and environmental benefits are balanced on a monetary basis, as the renaturation measures are monetized and used to compensate the monetized LCA results, e.g., of a product, organization or individual. In a case study, the implementation of the approach is presented to show the practical implementation of the CEC. The challenges of CEC include the integration of further impact categories, the availability of up-to-date and reliable monetization methods, the asynchrony and time-lag of the compensation from an ecosystem and biodiversity perspective and the proof of cost-efficiency of the renaturation measures. It is further discussed, if CEC can be a step beyond "climate neutrality" towards "environmental neutrality". The proposed approach should be further tested and is intended to foster progress in more comprehensive and robust offsetting of environmental impacts beyond climate change.

Cloning and Characterization of Two Novel PR4 Genes from Picea asperata.

Pathogenesis-related (PR) proteins are important in plant pathogenic resistance and comprise 17 families, including the PR4 family, with antifungal and anti-pathogenic functions. PR4 proteins contain a C-terminal Barwin domain and are divided into Classes I and II based on the presence of an N-terminal chitin-binding domain (CBD). This study is the first to isolate two PR4 genes, PaPR4-a and PaPR4-b, from Picea asperata, encoding PaPR4-a and PaPR4-b, respectively. Sequence analyses suggested that they were Class II proteins, owing to the presence of an N-terminal signal peptide and a C-terminal Barwin domain, but no CBD. Tertiary structure analyses using the Barwin-like protein of papaya as a template revealed structural similarity, and therefore, functional similarity between the proteins. Predictive results revealed an N-terminal transmembrane domain, and subcellular localization studies confirmed its location on cell membrane and nuclei. Real-time quantitative PCR (RT-qPCR) demonstrated that PaPR4-a and PaPR4-b expression levels were upregulated following infection with Lophodermium piceae. Additionally, PaPR4-a and PaPR4-b were induced in Escherichia coli, where the recombinant proteins existed in inclusion bodies. The renatured purified proteins showed antifungal activity. Furthermore, transgenic tobacco overexpressing PaPR4-a and PaPR4-b exhibited improved resistance to fungal infection. The study can provide a basis for further molecular mechanistic insights into PR4-induced defense responses.

On the emerging of thawing drip: Role of myofibrillar protein renaturation.

This study aimed to facilitate the understanding on the origin of thawing drip under different freezing rate. Eventually we observed significantly greater thaw loss produced by slow freezing (8.58%) as compared to fast freezing (6.41%) after 24 h of thawing. Back to the freezing, ice crystallization induced decline in pH and the cold denaturation of myofibrillar protein. However, independent of freezing rate, we noticed protein renaturation with pH restoring during thawing, evidenced by the decreasing surface hydrophobicity, increasing solubility and thermal stability, and gradually stabilized secondary structure. Meanwhile, the water-holding of myofibrils increased with thawing process along with the rising water mobility. Under fast freezing, the results indicated less extensive protein cold denaturation and lower water mobility during thawing. Besides, we proposed that the microenvironment of lower ionic strength in fast freezing should benefit the protein renaturation and water re-absorption, ultimately contributed to lower thaw loss.

Recombinant expression, purification, and characterization of full-length human BST-2 from Escherichia coli.

HIV-1 virus release from infected cells is blocked by human BST-2, but HIV-1 Vpu efficiently antagonises BST-2 due to direct transmembrane domain interactions that occur between each protein. Targeting the interaction between these two proteins is seen as viable for HIV-1 antiviral intervention. This study describes the successful over-expression and purification of a recombinant full-length human BST-2 from inclusion bodies using affinity and anion exchange chromatography. Two milligrams of purified full-length BST-2 were produced per litre of BL21 (DE3) T7 Express® pLysY E. coli culture. Far-UV circular dichroism validated the renaturing of the recombinant protein and retention of its secondary structure. Furthermore, through ELISA, a known human BST-2 binding partner, HIV-1 Vpu, was shown to bind to the renatured and purified protein, further validating its folding. To our knowledge this is the first report of the purification of a wild-type, full-length human BST-2 from Escherichia coli.

Assessment of renaturation measures for improvements in ecosystem services and flood risk mitigation.

The present paper offers a contribution to the research on social acceptance of interventions aimed at water ecosystem improvement and flood risk mitigation through renaturation measures. A CE study has been implemented to assess trade-offs between attributes of alternative projects, including social costs deriving from proposed actions of renaturation of river flows. The aim of our approach is to investigate the role of attitudinal factors in the valuation of costs and benefits generated by renaturation measures. A Hybrid Latent Class (HLC) model is applied to the data, revealing the existence of two distinct groups, characterised by different valuations of the attributes of the project. It is found that class membership depends on latent attitudes toward environmental protection and risk perception. Our study confirms the fruitfulness of the HLC modelling approach in stated preference studies regarding ecosystems valuation, as it provides a richer understanding of public preferences and allows more finely targeted policy indications.

[Cloning and expression of recombinant truncated SElX protein and evaluation on the related emetic activities].

Objective: To analyze the amino acid polymorphism of truncated Staphylococcal enterotoxin-like toxin X (tSElX), and to evaluate its related emetic activities. Methods: Sequence of tselx was compared with both the genome sequence of 145 CC398 strains completed in our research group and the NCBI database. Primers were designed to amplify the target gene of tselx, and the fragment was recombined into pMD18-T vector and sequenced. PCR product was digested with BamHⅠ and EcoRⅠ, and constructed into plasmid pGEX-6P-1 and pET-28a (+). After recombinant plasmid was identified, the protein expression was induced by IPTG. Proteins expressed in the form of inclusion bodies were denatured and renatured, then purified by affinity chromatography and ultrafiltration. Purified tSElX protein was then fed to common marmosets with the dose of 250 µg/kg to observe the vomiting reaction. Results: tselx gene was present in 145 strains of CC398 strains from the different origins (patients, healthy people and animals) in China. Homology of the amino acid sequence of the protein from the Chinese strains appeared 100.0%, while the homology with the four American strains were 97.8%(1) and 98.9%(3), respectively. Through two sets of expression systems and different induction conditions, tSElX was expressed in the form of inclusion bodies. The high purity soluble recombinant tSElX was thus obtained by denaturated and renaturated processes. At the dose of 250 µg/kg, tSElX protein did not cause vomiting in common marmosets. Conclusions: Results of this study showed that the amino acid sequence of tSElX was highly conserved and was universally present in a particular clone group. We obtained soluble recombinant tSElX protein with high purity. We also noticed that tSElX did not have the animal emetic activity at a dose of 250 µg/kg.

Optimizing a production strategy for a nonspecific nuclease from Yersinia enterocolitica subsp. palearctica in genetically engineered Escherichia coli.

A nuclease from Yersinia enterocolitica subsp. palearctica (Nucyep) is a newly found thermostable nonspecific nuclease. The heat-resisting ability of this nuclease would be extremely useful in biological research or pharmaceutical production. However, the application of this nuclease is limited because of its poor yield. This research aimed to improve Nucyep productivity by producing a novel genetically engineered Escherichia coli and optimizing the production procedures. After 4 h of induction by lactose, the new genetically engineered E. coli can express a substantial amount of Nucyep in the form of inclusion bodies. The yield was approximately 0.3 g of inclusion bodies in 1 g of bacterial pellets. The inclusion bodies were extracted by sonication and solubilized in an 8 M urea buffer. Protein renaturation was successfully achieved by dilution method. Pure enzyme was obtained after subjecting the protein solution to anion exchange. The Nucyep showed its nonspecific and heat resistant properties as previously reported (Boissinot et  al. 2016). Through a quantification method, its activity was determined to be 1.3 × 10 6 Kunitz units (K.U.)/mg. These results can serve as a reference for increasing Nucyep production.

Denaturation studies of Clarias gariepinus glutathione transferase in dilute and crowded solutions.

It is important to understand the effect of crowding conditions on the native structure and functional state of enzymes. Equilibrium denaturation studies of Clarius gariepinus GST (CgGST) by guanidine hydrochloride (GdHCl) under dilute conditions and in separate solutions of 0-100 g dm-3 Ficoll 70, polyethylene glycol 6000 (PEG 6000) and equal w/v mixtures of the two polymers at 25 °C and pH 7.4 were studied fluorometrically. The data were analyzed based on a two-state model assuming the native protein dimer separates into two monomers and then unfolds. The standard free energy of unfolding (ΔG°UN) increases with increasing concentration of each crowding agent in a manner suggesting that high concentrations of PEG 6000 and Ficoll 70 favour the native CgGST relative to the unfolded form. Ficoll 70 stabilizes the native CgGST better than PEG 6000 at low w/v concentration. A mixture of equal g/cm3 concentrations of both crowding agents, however, stabilizes the native form more effectively than either Ficoll 70 or PEG 6000 at equivalent w/v total concentration and is less sensitive to GdHCl. This is in strong agreement with the results of refolding studies, and suggests that a mixture of molecular crowders of widely different molecular weights might show enhanced excluded volume effects compared to a single crowder. Thus, mixed crowding agents more effectively protect the enzyme against denaturation and assist in renaturation better than a single crowder. This suggests a heterogeneous solution of crowders, as will be found within cells, enhances the beneficial effect of crowding on the folded protein stability.

An efficient large-scale refolding technique for recovering biologically active recombinant human FGF-21 from inclusion bodies.

Fibroblast growth factor 21 (FGF-21) is an important regulator in glycolipid metabolism that is a promising drug candidate for treatment of diabetes and obesity. However, the productivity of recombinant hFGF-21 (rhFGF-21) in Escherichia coli (E. coli) is relatively low, which limits its clinical application. To meet the clinical demand and control the production cost, rhFGF-21 proteins were expressed in inclusion bodies (IBs) form in Rosetta (DE3) by high cell density fermentation in 50-L scale. Hollow fiber membrane filtration technology was used to enrich the bacteria, wash, denature and refold the IBs in the current report. The renatured proteins were purified by two-step affinity chromatography. Authenticity of the purified rhFGF-21 was confirmed by the N-and C-terminal sequence, disulfide bond composition and molecular weight analyses. Results showed that the average target protein and recovery of rhFGF-21 expressed in IBs form of three batches were more than those of the soluble form. Both the rhFGF-21 proteins from the two forms showed equal potency in improving the glucose uptake in HepG2 cells and anti-diabetic effect in db/db mice. In this study, an efficient method for preparation of FGF-21 was established. This novel process provides an important technical basis for the large-scale production of rhFGF-21.

Purification of insoluble GST-fused and GST-cleaved Cav1.2 channel fragment by denaturation and renaturation.

Both recombinant glutathione-S-transferase (GST)-fused and GST-cleaved fragments of an L-type voltage-gated Ca2+ channel (Cav1.2) are used frequently in GST pull-down assays to investigate the interactions between regulatory proteins and the Cav1.2 channel. However, GST-fused and GST-cleaved proximal C-terminal fragments of the guinea-pig cardiac Cav1.2 channel (CT1, amino acids 1509-1791) heterologously expressed in Escherichia coli (E. coli) are difficult to be recovered in a bioactive form because they are only poorly soluble. In this study, we developed a new method to solubilize and purify CT1. GST-CT1 expressed in E. coli was extracted and treated with an inclusion body solubilization and renaturation kit. Then, after adsorption to glutathione Sepharose beads, GST-CT1 was treated with protease to release CT1. However, the cleaved CT1 was insoluble and remained attached to the beads. Therefore, CT1 was treated again with the inclusion body solubilization and renaturation kit. Using this method, GST-CT1 and CT1 were purified with a high yield. GST pull-down experiments showed a dose-dependent interaction between GST-CT1 and calmodulin (CaM), and between GST-CaM and CT1, suggesting recovered bioactivity of GST-CT1 and CT1. This protocol may also be applied to purify other insoluble GST-fused proteins.