Enhanced soluble expression of active recombinant human interleukin-29 using champion pET SUMO system

Munir, Ayesha; Ahmed, Nadeem; Akram, Muhammad; Fujimura, Naoki; Tahir, Saad; Malik, Kausar

doi:10.1007/s10529-023-03402-x

Cited by 5 publications

(2 citation statements)

References 47 publications

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“…Therefore, it does not obliterate the protein partner [ 16 ]. In addition, it cleaves at the C-terminus of the SUMO sequence, leaving no exogenous residues on the cleaved protein [ 23 ]. The SUMO-tag was cleaved without nonspecific proteolysis in this study.…”

Section: Discussionmentioning

confidence: 99%

Small ubiquitin-like modifier-tag and modified protein purification significantly increase the quality and quantity of recombinant African swine fever virus p30 protein

Chootip,

Hansoongnern,

Thangthamniyom

et al. 2024

Vet World

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Background and Aim: African swine fever (ASF) is a highly virulent and contagious viral disease caused by the ASF virus (ASFV). It has a significant impact on swine production throughout the world, while existing vaccines and specific treatments remain ineffective. ASFV p30 is a potent antigenic protein that induces protective antibodies immediately after infection; however, most recombinant p30 is insoluble. This study aimed to improve the solubility, yield, and purity of recombinant p30 by tagging it with a small ubiquitin-like modifier (SUMO) and modifying the protein purification process. Materials and Methods: SUMO fused with ASFV p30 (SUMO-p30) and p30 alone were cloned and expressed in Escherichia coli. SUMO-p30 and p30 solubility and expression levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein purification was modified by combining ammonium sulfate precipitation method with affinity chromatography. In addition, large-scale production of all versions of p30 were compared using SDS-PAGE and western blotting, and the purified p30 was used to develop the indirect enzyme-linked immunosorbent assay (ELISA). Results: The solubility and expression levels of SUMO-p30 were dramatically enhanced compared with that of p30. Modification of the purification process significantly increased purified and soluble SUMO-p30 and p30 yields by 6.59 and 1.02 μg/mL, respectively. Large-scale production confirmed that this procedure increased the quantity of recombinant p30 while maintaining protein purity and immunogenicity. The p30-based indirect ELISA was able to discriminate between positive and negative serum samples with statistically significant differences in mean optical density 450 values (p < 0.001). Conclusion: This study demonstrates the enhancement of solubility, purity, and yield of ASFV p30 expressed in E.coli by SUMO fusion tagging and combining ammonium sulfate precipitation with affinity chromatography for protein purification. These positive effects were sustained in large-scale production. Cleavage and removal of hexahistidine-SUMO tag from the fusion protein by protease may not be suitable when handling a large amount of the protein. However, the SUMO-fused p30 retained strong immunoreactivity to convalescent swine serum, indicating its application in immunization and diagnostic purposes. The expression and purification procedures in this study could be applied to increase solubility, quality, and quantity of other recombinant proteins as well. Keywords: African swine fever, diagnosis, indirect enzyme-linked immunosorbent assay, p30, protein expression, purification.

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Section: Discussionmentioning

confidence: 99%

Small ubiquitin-like modifier-tag and modified protein purification significantly increase the quality and quantity of recombinant African swine fever virus p30 protein

Chootip,

Hansoongnern,

Thangthamniyom

et al. 2024

Vet World

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“…SUMO tag in E. coli enables high expression of the target protein by correctly folding it (Butt et al 2005). In addition, SUMO fusion not only elevates the expression of the target protein, but also enhances the soluble form of its expression (Munir et al 2023;Takahashi et al 2023). However, no soluble expression of SM nuclease after SUMO fusion was observed in this study under the same expression conditions.…”

Section: Discussionmentioning

confidence: 99%

Expression and purification of inclusion body of Serratia marcescens nuclease relying on SUMO fusion

Zang,

Wei,

Deng

et al. 2023

Preprint

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Serratia marcescens nuclease (SM nuclease) can remove nucleic acid residues in recombinant protein drugs and reduce the viscosity of bacteria, exhibiting great significance in investigating these drugs. However, its underexpression in E. coli leads to less protein amount obtained by purification of inclusion body and low enzyme activity. In this study, the Small Ubiquitin-like Modifier (SUMO) tag was fused to the N-terminus of the SM nuclease and cloned into the pET28a vector. Subsequently, the expression and purification of inclusion body of the SUMO-fused SM nuclease were compared with those of SM nuclease without SUMO fusion. The results revealed that SUMO fusion elevated the expression of inclusion body of the SM nuclease, but exerted no effect on soluble expression of the protein. Meanwhile, SUMO fusion increased the solubility of inclusion body proteins and enhanced the removal of surface impurities during inclusion body washing. On the other hand, SUMO fusion promoted correct folding of the protein and improved the efficiency of refolding. The High-Performance Liquid Chromatography (HPLC) results indicated a protein concentration of 99% after two cycles of affinity chromatography for SUMO-fused SM nuclease. Additionally, the activity of the SUMO-fused protein (4×106 U/mg) was 32 times higher than that of the unfused protein. SUMO fusion yielded approximately 10 mg of SM nuclease protein with a purity of 99% from 1 g of bacteria.

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Essential factors, advanced strategies, challenges, and approaches involved for efficient expression of recombinant proteins in Escherichia coli

Eskandari,

Nezhad,

Leow

et al. 2024

Arch Microbiol

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Enhanced soluble expression of active recombinant human interleukin-29 using champion pET SUMO system

Cited by 5 publications

References 47 publications

Small ubiquitin-like modifier-tag and modified protein purification significantly increase the quality and quantity of recombinant African swine fever virus p30 protein

Small ubiquitin-like modifier-tag and modified protein purification significantly increase the quality and quantity of recombinant African swine fever virus p30 protein

Expression and purification of inclusion body of Serratia marcescens nuclease relying on SUMO fusion

Essential factors, advanced strategies, challenges, and approaches involved for efficient expression of recombinant proteins in Escherichia coli

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