Cloning, High Expression and Purification of Recombinant Human Intereferon-β-1b in Escherichia coli

Rao, Dasari Venkata Krishna; Ramu, Chatadi Tulasi; Rao, Joginapally V.; Narasu, M. Lakshmi; Rao, Adibhatla Kali Satya Bhujanga

doi:10.1007/s12010-008-8318-9

Cited by 18 publications

(11 citation statements)

References 44 publications

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“…Besides IL-10, the cytokines Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), Interleukin-5 (IL-5) and Interfernon-beta (IFN-β) are also described as 3D domain swapping proteins [9] and have been heterologously expressed in several platforms. Upon expression in E. coli the formation of inclusion bodies were described for these four cytokines [25]–[28]. Although formation of inclusion bodies could be indicative of multimerisation, they are a relatively common feature when expressing eukaryotic proteins in E. coli due to aggregation of folding intermediates.…”

Section: Discussionmentioning

confidence: 99%

“…Although formation of inclusion bodies could be indicative of multimerisation, they are a relatively common feature when expressing eukaryotic proteins in E. coli due to aggregation of folding intermediates. However, in the case of IFN-β and IL-5, experiments also demonstrated the need for slow removal of denaturing agents to prevent aggregation to reoccur after solubilisation of inclusion bodies [25], [26]. To our knowledge, IL-5 has not been produced in plants before and expression of human IFN-β in lettuce leaves resulted in poor yields [29].…”

Section: Discussionmentioning

confidence: 99%

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3D Domain Swapping Causes Extensive Multimerisation of Human Interleukin-10 When Expressed In Planta

et al. 2012

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Heterologous expression platforms of biopharmaceutical proteins have been significantly improved over the last decade. Further improvement can be established by examining the intrinsic properties of proteins. Interleukin-10 (IL-10) is an anti-inflammatory cytokine with a short half-life that plays an important role in re-establishing immune homeostasis. This homodimeric protein of 36 kDa has significant therapeutic potential to treat inflammatory and autoimmune diseases. In this study we show that the major production bottleneck of human IL-10 is not protein instability as previously suggested, but extensive multimerisation due to its intrinsic 3D domain swapping characteristic. Extensive multimerisation of human IL-10 could be visualised as granules in planta. On the other hand, mouse IL-10 hardly multimerised, which could be largely attributed to its glycosylation. By introducing a short glycine-serine-linker between the fourth and fifth alpha helix of human IL-10 a stable monomeric form of IL-10 (hIL-10mono) was created that no longer multimerised and increased yield up to 20-fold. However, hIL-10mono no longer had the ability to reduce pro-inflammatory cytokine secretion from lipopolysaccharide-stimulated macrophages. Forcing dimerisation restored biological activity. This was achieved by fusing human IL-10mono to the C-terminal end of constant domains 2 and 3 of human immunoglobulin A (Fcα), a natural dimer. Stable dimeric forms of IL-10, like Fcα-IL-10, may not only be a better format for improved production, but also a more suitable format for medical applications.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

3D Domain Swapping Causes Extensive Multimerisation of Human Interleukin-10 When Expressed In Planta

et al. 2012

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“…Already has used sodium deoxycholate and Triton X-100 for removing membrane proteases (39). Dasari also showed that the increase in the concentration of Triton X-100 causes IBs to precipitate via increasing buffer viscosity (40). In this research, the best efficiency of IBs recovery was found at a concentration 0.5 M of Triton X-100 (without the use of Sodium DeoxyCholate in the second phase of the washing stage).…”

Section: Discussionmentioning

confidence: 62%

Optimizing Primary Recovery and Refolding of Human Interferon-b from Escherichia coli Inclusion Bodies

Farzaneh¹,

Khodabandeh²,

Arpanaei³

et al. 2014

Iran J Biotech

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Background:The refolding of proteins from inclusion bodies is affected by several factors, including solubilization of inclusion bodies by denaturants, removal of the denaturant, and assistance of refolding by small molecule additives. Objectives: The purpose of this study was optimization of recombinant human interferon-β purification in order to achieve higher efficiency, yield, and a product with a better and more suitable biological activity. Materials and Methods: Triton X-100 and sodium deoxycholate were used to wash the recombinant human interferon-β inclusion bodies prior to solubilization. The inclusion bodies solubilization process was performed by denaturants and reducing agents; guanidine-hydrochloride, urea, β-mercaptoethanol and dithiothritol. Results: The best recovery was obtained in the presence of 0.5% TritonX-100 (v/v). Low concentrations of urea only gave a marginal improvement on the refolding of recombinant human interferon-β. Successful refolding was achieved by gradient elution (decreasing the guanidine-hydrochloride concentration) in the presence of L-arginine. Partial purification was also achieved continuously, and recombinant human interferon-β was recovered with 93.5% purity. The interferon prepared in this project was biologically active and inhibited the replication of vesicular stomatitis virus in Hela cells, when compared to the standard interferon. Conclusions: In this research, the best recovery of inclusion bodies was found at a concentration 0.5 M of Triton X-100, the maximum efficiency of solubility was found in pH 10.5 and the maximum efficiency of refolding was achieved by final buffer containing 2M urea and 0.6 M L-Arg.

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“…Escherichia coli is a well‐known host for the effective expression of nonglycosylated recombinant proteins . However, the high‐level expression of eukaryotic proteins in E. coli often leads to the formation of insoluble protein aggregates in the cytoplasm, referred to as inclusion bodies (IBs) .…”

Section: Introductionmentioning

confidence: 99%

“…Due to its nature, the solubilization of recombinant proteins expressed as IBs—derived from partially folded intermediates, lipids, carbohydrates, DNA, RNA, and other cellular proteins —is a challenging task. Frequently, IBs are treated with denaturing agents along with detergents and reducing agents to achieve high protein solubilization , considering the requirements of their further processing with the simplest execution.…”

Section: Introductionmentioning

confidence: 99%

Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies

et al. 2013

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In this work, a high-resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single-component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon-β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r(2) ) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon-β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines.

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Cloning, High Expression and Purification of Recombinant Human Intereferon-β-1b in Escherichia coli

Cited by 18 publications

References 44 publications

3D Domain Swapping Causes Extensive Multimerisation of Human Interleukin-10 When Expressed In Planta

3D Domain Swapping Causes Extensive Multimerisation of Human Interleukin-10 When Expressed In Planta

Optimizing Primary Recovery and Refolding of Human Interferon-b from Escherichia coli Inclusion Bodies

Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies

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