Novel peptide inhibitor of human tumor necrosis factor-α has antiarthritic activity.

The inhibition of tumor necrosis factor (TNF)-α trimer formation renders it inactive for binding to its receptors, thus mitigating the vicious cycle of inflammation. We designed a peptide (PIYLGGVFQ) that simulates a sequence strand of human TNFα monomer using a series of in silico methods, such as active site finding (Acsite), protein-protein interaction (PPI), docking studies (GOLD and Flex-X) followed by molecular dynamics (MD) simulation studies. The MD studies confirmed the intermolecular interaction of the peptide with the TNFα. Fluorescence-activated cell sorting and fluorescence microscopy revealed that the peptide effectively inhibited the binding of TNF to the cell surface receptors. The cell culture assays showed that the peptide significantly inhibited the TNFα-mediated cell death. In addition, the nuclear translocation of the nuclear factor kappa B (NFκB) was significantly suppressed in the peptide-treated A549 cells, as observed in immunofluorescence and gel mobility-shift assays. Furthermore, the peptide protected against joint damage in the collagen-induced arthritis (CIA) mouse model, as revealed in the micro focal-CT scans. In conclusion, this TNFα antagonist would be helpful for the prevention and repair of inflammatory bone destruction and subsequent loss in the mouse model of CIA as well as human rheumatoid arthritis (RA) patients. This calls upon further clinical investigation to utilize its potential effect as an antiarthritic drug.

Dominant B cell-T cell epitopes instigated robust immune response in-silico against Scrub Typhus.

Scrub typhus, a potentially life-threatening infectious disease, is attributed to bacteria Orientia tsutsugamushi (O. tsutsugamushi). The transmission of this illness to humans occurs through the bite of infected chiggers, which are the larval forms of mites belonging to the genus Leptotrombidium. In this research, we developed a subunit vaccine specifically designed to target outer membrane proteins. Immunodominant cytotoxic T-lymphocytes (CTLs), B- lymphocytes (BCLs), and major histocompatibility complex (MHC)- II epitopes were identified using machine learning and bioinformatics approaches. These epitopes were arranged in different combinations with the help of suitable linkers like AAY, KK, GPGPG and adjuvant (cholera toxin B) that resulted in a vaccine construct. Physiochemical properties were assessed, where the predicted solubility (0.571) was higher than threshold value. Tertiary structure was predicted using I-TASSER web server and evaluated using Ramachandran plot (94 % residues in most favourable region) and z-score (-6.04), which had shown the structure to have good stability and residue arrangement. Molecular docking with immune receptors, Toll-like receptor (TLR)-2 and -4 showed good residue interaction with 13 and 5 hydrogen bonds respectively. Molecular dynamics simulations of receptor-ligand complex provided the idea about the strong interaction having 1.524751 × 10-5 eigenvalue. Amino acid sequence of vaccine was converted to nucleotide sequence and underwent codon optimization. The optimized codon sequence was used for in-silico cloning, which provided idea about the possibility of synthesis of vaccine using E. coli as host. Overall, this study provided a promising blueprint for a scrub typhus vaccine, although experimental validation is needed for confirmation. Furthermore, it is crucial to acknowledge that while bioinformatics provides valuable insights, in-vitro and in-vivo studies are imperative for a comprehensive evaluation of vaccine candidate. Thus, the integration of computational predictions with empirical research is essential to validate the efficacy, safety, and real-world applicability of the designed vaccine against Scrub Typhus. Nevertheless, the findings are good to carry forward for in-vitro and in-vivo investigations.

Excellent Adsorption of Lead (II) and Chromium (VI) from Water Using Zwitterions (-NH3+ and -COO-) Functionalized Nano Lanthanum Oxide: Kinetic, Isotherm, Thermodynamic, and Surface Mechanism.

Zwitterion amino acid l-cysteine functionalized lanthanum oxide nanoparticles (l-Cyst-La2O3 NPs) have been synthesized for the first time with lanthanum acetate as the precursor, NH4OH as the base, and l-cysteine as the in situ functionalized mediator. The typical size of l-Cyst-La2O3 NPs was obtained in the range of 15-20 nm from the TEM technique. A cytotoxicity test of l-Cyst-La2O3 NPs was performed in Raw 264.7 cell lines, which were shown to be highly biocompatible. The point zero charge pH (pHPZC) of bare and l-Cyst functionalized La2O3 NPs was obtained at pH 6 and 2. The maximum uptake capacities of l-Cyst-La2O3 NPs at temperatures 25-45 °C were obtained as 137-282 mg/g for Pb2+ and 186-256 mg/g for Cr6+. All of these values are much higher than those reported in the literature with other nanomaterials. The presence of -SH, -NH2, and -COOH functional groups in zwitterion l-cysteine provides multiple binding sites leading to the high adsorption of Pb2+ and Cr6+. Five-cycle desorption studies were successfully performed to regenerate the spent l-Cyst-La2O3 NPs.

Stimuli-responsive magnetic silica-poly-lactic-co-glycolic acid hybrid nanoparticles for targeted cancer chemo-immunotherapy.

Chemotherapy and immunotherapy are two important modalities in cancer management. However, due to multiple reasons, a monotherapy is only partially effective. Hence, if used concurrently in targeted and stimuli-responsive manner, it could have been superior therapeutically. To facilitate co-delivery of chemotherapeutic and immunotherapeutic agent to the target cancer cells, engineered nanoparticles, i.e., a pH-responsive polymer PLGA-coated magnetic silica nanoparticles (Fe3O4-SiO2-PLGA-PDA-PTX-siRNA NPs) encapsulating paclitaxel (PTX) and siRNA against programmed cell death ligand-1 (PD-L1) are synthesized and characterized. Developed nanoparticles demonstrated pH-sensitive sustained drug release up to 10 days. In vitro 4T1 cell line studies showed efficient cellular uptake, PD-L1 gene downregulation, and apoptosis. Further, in vivo efficacy studies carried out in the mice model demonstrated a significant reduction of tumor growth following treatment with dual-Fe3O4-SiO2-PLGA-PDA-PTX-siRNA NPs as compared with monotherapy with Fe3O4-SiO2-PLGA-PDA-PTX NPs. The high therapeutic efficacy observed with dual-Fe3O4-SiO2-PLGA-PDA-PTX-siRNA NPs was mainly due to the cytotoxic effect of PTX combined with targeted silencing of the gene of interest, i.e., PD-L1, which in turn improve CD8+ T cell-mediated cancer cell death as evident with increased proliferation of CD8+ T cells in co-culture experiments. Thereby, dual-Fe3O4-SiO2-PLGA-PDA-PTX-siRNA NPs may have a promising anti-cancer treatment potential against breast cancer; however, the beneficial effects of dual loading of PTX + PD-L1 siRNA may be corroborated against other cancer models such as lung and colorectal cancer models as well as in clinical trials.

Beyond the polysaccharide and glycoconjugate vaccines for Streptococcus pneumoniae: Does protein/peptide nanovaccines hold promises?

Streptococcus pneumoniae having almost 98 serotypes and being common cause of acute otitis media, pneumonia, bacteremia, meningitis etc., which results in high mortality and morbidity globally. Although vaccines like PCV-13 and PPV-23 are available, some problems like serotype replacement and poor immunogenicity in children, old age and immunocompromised people has been observed. To overcome these drawbacks protein/peptide-based vaccine can be a good strategy as these provides wide serotype coverage. However, immunogenicity of protein subunit vaccines is lower, that issue can be solved by using adjuvants. Recently nanoparticles as an adjuvant for vaccine delivery being used, which has provided not only good immunogenicity but also improved delivery and efficiency of protein-based vaccines. In this review we have discussed the latest advancement of nanoparticles-based protein/peptide vaccine delivery for Streptococcus pneumoniae.

Electrochemical Detection of Vibrio cholerae by Amine Functionalized Biocompatible Gadolinium Oxide Nanoparticles.

Herein, we report the biocompatible amine-functionalized gadolinium oxide nanoparticles (Gd2O3 NPs) for the possibility of electrochemical detection of Vibrio cholerae (Vc) cells. The microwave irradiation process is applied to synthesize Gd2O3 NPs. The amine (NH2) functionalization is carried out via overnight stirring with 3(Aminopropyl)triethoxysilane (APTES) at 55 °C. The size of NPs amine functionalized APETS@Gd2O3 NPs are determined by transmission electron microscopy (TEM). APETS@Gd2O3 NPs are further electrophoretically deposited onto indium tin oxide (ITO) coated glass substrate to obtain working electrode surface. The monoclonal antibodies (anti-CT) specific to cholera toxin associated to Vc cells are covalently immobilized onto the above electrodes using EDC-NHS chemistry and further BSA is added to obtain the BSA/anti-CT/APETS@Gd2O3/ITO immunoelectrode. Further, this immunoelectrode shows the response for cells in CFU range from 3.125 × 106 to 30 × 106 and is very selective with sensitivity and LOD 5.07 mA CFUs mL cm-2 and 0.9375 × 106 CFU respectively. To establish a future potential for APTES@Gd2O3 NPs in field of biomedical applications and cytosensing, the effect of APTES@Gd2O3 NPs on mammalian cells is also observed using in vitro cytotoxicity assay and cell cycle analysis.

Immunogenicity Evaluation of Thermostable Microparticles Entrapping Receptor Binding Domain of SARS-CoV-2 by Single Point Administration.

Receptor binding domain (RBD) of SARS-CoV-2 is a prime vaccine target against which neutralizing antibody responses are directed. Purified RBD as a vaccine candidate warrants administration of multiple doses along with adjuvants and use of delivery systems to improve its immunogenicity. The present investigation examines the immunogenicity of RBD delivered by biodegradable polymer particles from single dose administration. Mice upon single point immunization of RBD entrapped microparticles generated improved antibody response. The polymer microparticles showed better temperature stability and could be stored at 37 degrees for one month without any considerable loss of immunogenicity. Further, immunization with microparticles could elicit memory antibody response upon challenge after four months of single dose administration. Thus, using microparticles entrapping RBD as a vaccine candidate confer improved immunogenicity, temperature stability and recall response. These thermostable microparticles seem to be a potentially cost-effective approach which can help in dose reduction, provide a wider access of vaccines and accelerate the end of global pandemic.

RBD decorated PLA nanoparticle admixture with aluminum hydroxide elicit robust and long lasting immune response against SARS-CoV-2.

Nanoparticles-based multivalent antigen display has the capability of mimicking natural virus infection characteristics, making it useful for eliciting potent long-lasting immune response. Several vaccines are developed against global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However these subunit vaccines use mammalian expression system, hence mass production with rapid pace is a bigger challenge. In contrast E. coli based subunit vaccine production circumvents these limitations. The objective of the present investigation was to develop nanoparticle vaccine with multivalent display of receptor binding domain (RBD) of SARS-CoV-2 expressed in E. coli. Results showed that RBD entrapped PLA (Poly lactic acid) nanoparticle in combination with aluminum hydroxide elicited 9-fold higher immune responses as compared to RBD adsorbed aluminum hydroxide, a common adjuvant used for human immunization. It was interesting to note that RBD entrapped PLA nanoparticle with aluminum hydroxide not only generated robust and long-lasting antibody response but also provided Th1 and Th2 balanced immune response. Moreover, challenge with 1 µg of RBD alone was able to generate secondary antibody response, suggesting that immunization with RBD-PLA nanoparticles has the ability to elicit memory antibody against RBD. Plaque assay revealed that the antibody generated using the polymeric formulation was able to neutralize SARS-CoV-2. The RBD entrapped PLA nanoparticles blended with aluminum hydroxide thus has potential to develop asa subunit vaccine against COVID-19.

Troxerutin-Mediated Complement Pathway Inhibition is a Disease-Modifying Treatment for Inflammatory Arthritis.

Troxerutin (TXR) is a phytochemical reported to possess anti-inflammatory and hepatoprotective effects. In this study, we aimed to exploit the antiarthritic properties of TXR using an adjuvant-induced arthritic (AIA) rat model. AIA-induced rats showed the highest arthritis score at the disease onset and by oral administration of TXR (50, 100, and 200 mg/kg body weight), reduced to basal level in a dose-dependent manner. Isobaric tags for relative and absolute quantitative (iTRAQ) proteomics tool were employed to identify deregulated joint homogenate proteins in AIA and TXR-treated rats to decipher the probable mechanism of TXR action in arthritis. iTRAQ analysis identified a set of 434 proteins with 65 deregulated proteins (log2 case/control≥1.5) in AIA. Expressions of a set of important proteins (AAT, T-kininogen, vimentin, desmin, and nucleophosmin) that could classify AIA from the healthy ones were validated using Western blot analysis. The Western blot data corroborated proteomics findings. In silico protein-protein interaction study of tissue-proteome revealed that complement component 9 (C9), the major building blocks of the membrane attack complex (MAC) responsible for sterile inflammation, get perturbed in AIA. Our dosimetry study suggests that a TXR dose of 200 mg/kg body weight for 15 days is sufficient to bring the arthritis score to basal levels in AIA rats. We have shown the importance of TXR as an antiarthritic agent in the AIA model and after additional investigation, its arthritic ameliorating properties could be exploited for clinical usability.

Contraceptive efficacy of recombinant porcine zona proteins and fusion protein encompassing canine ZP3 fragment and GnRH in female beagle dogs.

PROBLEM: To manage population of dogs (Canis familiaris), the efficacy of recombinant proteins-based contraceptive vaccines to inhibit fertility has been evaluated in female beagle dogs. METHOD OF STUDY: Female beagle dogs (n = 4) were immunized with physical mixture of Escherichia coli-expressed recombinant porcine ZP3 with promiscuous T cell epitope of tetanus toxoid (TT-KK-pZP3) and porcine ZP4 with promiscuous T cell epitope of bovine RNase (bRNase-KK-pZP4), or with a fusion protein encompassing dog ZP3 fragment and two copies of GnRH with appropriate promiscuous T cell epitopes (dZP3-GnRH2 ); control animals received only alum, the adjuvant. The immunized animals were followed-up for antibody titres by ELISA as well as for fertility status subsequent to mating with male dogs. RESULTS: Active immunization of female dogs following a three injections schedule at 4-week intervals with a physical mixture of TT-KK-pZP3 + bRNase-KK-pZP4 as well as dZP3-GnRH2 , led to generation of significant antibody titres against respective recombinant proteins. Active immunization with dZP3-GnRH2 also led to generation of antibodies reactive with both dZP3 and GnRH. A booster dose on day 383 led to an increase in antibody titres and circulating antibodies against respective recombinant proteins could be observed on day 528. Antibodies in immune serum samples from dogs immunized with TT-KK-pZP3 + bRNase-KK-pZP4 or dZP3-GnRH2 reacted with native canine ZP as assessed by an indirect immunofluorescence assay. Mating studies revealed a reduced number of pregnancies as well as a significant reduction in the number of pups born in the female dogs immunized with dZP3-GnRH2 as compared to the adjuvanted control. Curtailment of pregnancy in dZP3-GnRH2 immunized group was associated with antibody titres against dZP3-GnRH2 . However, immunization with recombinant TT-KK-pZP3 + bRNase-KK-pZP4 did not significantly decrease the number of pups born as compared to the adjuvanted control. CONCLUSION: These studies revealed the potential of recombinant dZP3-GnRH2 -based contraceptive vaccine to curtail fertility in female dogs. Large scale studies to establish the efficacy and safety of this recombinant protein for the management of community dog population are thus warranted.

Microparticles entrapping pneumococcal protein SP0845 show improved immunogenicity and temperature stability.

Protein based vaccines are the most safe and affordable strategy to combat pneumococcal disease circumventing the limitations of conventional polysaccharide-based vaccines like serotype dependence, high cost and inability to be administered to immunocompromised. SP0845 is a highly conserved vaccine candidate shown to provide protection against heterologous strains of Streptococcus pneumoniae, the primal cause of pneumonia. However, the associated poor immunogenicity warrants the need for adjuvants and multiple doses to mount desired responses. The present study relates to improve the immunogenicity of pneumococcal protein SP0845 by use of poly lactic acid biodegradable polymer microparticles. The immunization studies showed that microparticles elicited higher antibody response compared to alum adjuvanted protein and this immunopotentiation was achieved without the use of any additional adjuvant. They were also capable of eliciting secondary antibody response upon boosting after four months. Further, the particles upon storage at 25 and 37 °C for one month were still capable of mounting an immune response equivalent to those stored in cold chain. Thus, using microparticles entrapping SP0845 for immunization not only improve the immunogenicity but also offer better temperature stability. This can greatly reduce the cost and increase access of protein-based vaccine to resource limited settings.

Solubilization and Refolding of Inclusion Body Proteins.

Expression of heterologous proteins in E. coli often leads to the formation of protein aggregates known as inclusion bodies (IBs). Inclusion body aggregates pose a major hurdle in the recovery of bioactive proteins from E. coli. Usage of strong denaturing buffers for solubilization of bacterial IBs results in poor recovery of bioactive protein. Structure-function understanding of IBs in the last two decades have led to the development of several mild solubilization buffers, which improve the recovery of bioactive from IBs. Recently, combinatorial mild solubilization methods have paved the way for solubilization of wide range of inclusion bodies with appreciable refolding yield. Here, we describe a simple protocol for solubilization and refolding of an inclusion body protein with appreciable recovery.

Solubilization and refolding of variety of inclusion body proteins using a novel formulation.

Formation of protein aggregates as inclusion bodies (IBs) still poses a major hurdle in the recovery of bioactive proteins from E. coli. Despite the development of many mild solubilization buffers in last two decades, high-throughput recovery of functional protein from wide range of IBs is still a challenge at an academic and industrial scale. Herein, a novel formulation for improved recovery of bioactive protein from variety of bacterial IBs is developed. This novel formulation is comprised of 20% trifluoroethanol, 20% n-propanol and 2 M urea at pH 12.5 which disrupts the major dominant forces involved in protein aggregation. An extensive comparative study of novel formulation conducted on different IBs demonstrates its high solubilization and refolding efficiency. The overall yield of bioactive protein from human growth hormone expressed as bacterial IBs is reported to be around 50%. This is attributed to the capability of novel formulation to disrupt the tertiary structure of the protein while protecting the secondary structure of the protein, thereby reducing the formation of soluble aggregates during refolding. Thus, the formulation can eliminate the need of screening and optimizing various solubilization formulation and will improve the efficiency of recovering bioactive protein from variety of IB aggregates.

Cellular uptake of polylactide particles induces size dependent cytoskeletal remodeling in antigen presenting cells.

Phagocytosis of particulate vaccine delivery systems is a critical immune mechanism involved in antigen capture and processing by macrophages and dendritic cells. The internalization and degradation of the particles involve a complex sequence of events. This process coordinates lipids, signaling proteins, and the cytoskeleton. Dynamic changes in the actin cytoskeleton are essential for phagocytosis and antigen presentation. Knowledge regarding the correlation of surface properties, attached ligand density and geometric size of particles with the efficiency of phagocytosis may facilitate their design and application. To investigate this, polylactide biodegradable particles with different diameters (2-4 µm and 200-300 nm) were exposed to murine macrophages and dendritic cells and the effect of size on a series of cellular responses was evaluated. Cellular uptake studies using microscopy and flow cytometry showed size dependent internalization of particles, with nanoparticles accumulating in cells at a faster rate. The particles induced homoaggregation of cells and also showed cytoskeletal remodeling that could be inhibited by cytochalasin-D. Scanning electron microscopy images showed the time dependent formation of phagocytic cups and invaginations that promote particle uptake. The particles were observed to co-localized with the endo-lysosomal compartments after phagocyotosis. In our experiments, particle mediated immunoactivation, antigen processing and cytokine secretion have shown a good correlation with the uptake process. These findings would allow a better understanding of the process of particle uptake and may be instrumental in the rational design of optimal vaccine delivery systems.

Molecular Attributes Associated With Refolding of Inclusion Body Proteins Using the Freeze-Thaw Method.

Understanding the structure-function of inclusion bodies (IBs) in the last two decades has led to the development of several mild solubilization buffers for the improved recovery of bioactive proteins. The recently developed freeze-thaw-based inclusion body protein solubilization method has received a great deal of attention due to its simplicity and cost-effectiveness. The present report investigates the reproducibility, efficiency, and plausible mechanism of the freeze-thaw-based IB solubilization. The percentage recovery of functionally active protein species of human growth hormone (hGH) and L-asparaginase from their IBs in Escherichia coli and the quality attributes associated with the freeze-thaw-based solubilization method were analyzed in detail. The overall yield of the purified hGH and L-asparaginase protein was found to be around 14 and 25%, respectively. Both purified proteins had functionally active species lower than that observed with commercial proteins. Biophysical and biochemical analyses revealed that the formation of soluble aggregates was a major limitation in the case of tough IB protein like hGH. On the other hand, the destabilization of soft IB protein like L-asparaginase led to the poor recovery of functionally active protein species. Our study provides insight into the advantages, disadvantages, and molecular-structural information associated with the freeze-thaw-based solubilization method.

Preparation and quality by design assisted (Qb-d) optimization of bioceramic loaded microspheres for periodontal delivery of doxycycline hyclate.

PLGA (Lactic- co-glycolic acid) coated chitosan microspheres loaded with hydroxyapatite and doxycycline hyclate complex were developed in the present study for periodontal delivery. A modified single emulsion method was adopted for the development of microspheres. Formulation was optimized on the basis of particle size, drug loading and encapsulation efficiency with the central composite design using 23 factorial design. Microspheres were optimized and electron microscopy revealed their spherical shape and porous nature. In-vitro study showed initial burst and then sustained release behavior of the formulation for 14 days. Further, in-vitro antibacterial study performed on E. coli (ATCC-25922) and S. aureus (ATCC-29213) revealed concentration dependent activity. Also, in-vitro cyto-toxicity assessment ensures biocompatibility of the formulation with the fibroblast's cells. Overall, the quality by design assisted PLGA microspheres, demonstrated the desired attributes and were found suitable for periodontal drug delivery.

Evaluation of size, shape, and charge effect on the biological interaction and cellular uptake of cerium oxide nanostructures.

Cerium oxide (CeO2) at the nanoscale has prolifically attracted the immense interest of researchers due to its switchable oxidation states (Ce3+/Ce4+) that play a crucial role in many biological activities. The present work reports the evaluation of size, shape, and charge effect on the biological interaction with RAW 264.7 cells for three nanostructures of CeO2(CeO2NS) namely nanocubes (NCs), nanorods (NRs), and nanoparticles (NPs). These NS exhibits similar composition and have average diameter values in the order of NCs < NRs â‰… NPs. The values of zeta potential revealed the anionic nature of NS with surface charge in order of NCs < NPs < NRs. The cellular interaction of CeO2NS was analyzed for cytotoxicity, cellular uptake, and morphological studies. Quantitative determination of the uptake of CeO2NS exhibited concentration-dependent uptake in the order as NCs > NPs > NRs. The proposed possible mechanisms of cellular uptake revealed that different structures tended to use the various endocytosis pathways in different proportions.

Protocol for in-vitro purification and refolding of hexachlorocyclohexane degrading enzyme haloalkane dehalogenase LinB from inclusion bodies.

LinB is an important haloalkane dehalogenase involved in the degradation pathway of different isomers of hexachlorocyclohexane (HCH), mainly in catalyzing degradation of the notorious ß-HCH. The HCH isomers are known to have neurotoxic, carcinogenic and estrogenic effects. Enzymatic bioremediation for decontamination of ß- as well as other HCH isomers can prove to be a potential remediation strategy. For any bioremediation technology that is to be developed, apart from having high turnover number, the candidate enzyme must also be available in sufficient amounts. In this direction, the LinB variants reported in database were tested in laboratory studies. The variant LinBSSO4-3 however could not be obtained in soluble fraction by using standard procedures. The protein LinBSSO4-3 was cloned in pDEST17 vector and codon optimized for better expression in Escherichia coli BL21AI using a strong T7 promoter. However, the over-expression of this protein in ectopic host E. coli, led to aggregation of the protein in form of inclusion bodies, which are insoluble aggregates of misfolded or partially folded proteins. SEM analysis of the inclusion bodies showed them as aggregated spherical particles. The inclusion bodies were isolated using high speed sonication and homogenization. This was followed by solubilization in the strong denaturing agent urea. Refolding into its native state was done by using pulsatile refolding. This was done by slowly decreasing the denaturant concentration in the presence of sucrose. The turnover number of the refolded protein was then determined for different isomers of HCH. The protein was found to have a turnover number of ∼43 molecules min-1 on ß-HCH and ∼13 molecules min-1 on δ-HCH. Additionally, a mutation I253 M in the active site of the enzyme was found to drastically decrease the enzyme activity on ß-HCH. Taking into consideration the wide range of substrates of haloalkane dehalogenases, such a protocol for inclusion body refolding will contribute to the field of bioremediation technology development for organochlorines, specifically HCH. Such a protocol for refolding of haloalkane dehalogenases from inclusion bodies has not been developed or reported before.