Expression and purification of the Lipl41, a surface-exposed lipoprotein antigen of pathogenic Leptospira spp.

Golab, Narges; Khaki, Pejvak; Harzandi, Naser; Esmaelizad, Majid; Tebianian, Majid

doi:10.24099/vet.arhiv.0803

Cited by 3 publications

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“…Based on the optimization results, the high yield of purified Loa22 protein showed that the optimized gene construct according to the genomic codon usage in the E. coli , is probably one of the reasons for the higher yield compared to restriction cloning with wild-type sequences. Previously, expression of LipL41 using a codon optimization approaches have been conducted and have obtained considerable amounts of recombinant protein, which is useful for academic studies and preclinical work ( 32 ).…”

Section: Discussionmentioning

confidence: 99%

Improvement the expression and purification of Loa22: a lipoprotein with OmpA domain from pathogenic Leptospira serovars

Gharakhani,

Ghasemi,

Khaki

et al. 2023

IJM

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Background and Objectives: One of the highly conserved outer membrane proteins expressed only by pathogenic Lepto- spires is Loa22. The study aims is to achieve the optimum conditions for high expression of recombinant Loa22 (rLoa22) protein. Materials and Methods: Complete coding sequence of loa22 gene sub-cloned into a pET32a (+) expression vector. BL21 competent E. coli (pLysS) used as expression host for transformation. The recombinant clones selected on ampicillin plates and subjected to PCR by using pET T7 primers. Then expression conditions optimized by adjusting parameters such as cul- ture media, induction time, temperature, and IPTG concentration. Results: SDS-PAGE analysis showed that high production of rLoa22 protein obtained when post induction incubation, IPTG concentration, and duration of induction were 37ºC, 0.1 M and 5 h in 2×TY medium respectively. The purification of rLoa22 protein under native conditions using Ni-NTA pull-down was optimum in one hour binding at 37°C, five times washing process and elution buffer with a pH 7.4 and a 0.3 M imidazole concentration. Conclusion: The findings of the study led to high production of pure Loa22 protein, which can form the basis for future investigation on the design of rapid diagnostic tests and more effective vaccine candidates for leptospirosis

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Section: Discussionmentioning

confidence: 99%

Improvement the expression and purification of Loa22: a lipoprotein with OmpA domain from pathogenic Leptospira serovars

Gharakhani,

Ghasemi,

Khaki

et al. 2023

IJM

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show abstract

“…Based on the optimization results, the high yield of puri ed Loa22 protein showed that the optimized gene construct according to the genomic codon usage in the E. coli, is probably one of the reasons for the higher yield compared to restriction cloning with wild-type sequences. Previously, expression of LipL41 by Golab et al 2020, and LipL21 by Kamalzadeh et al 2021, using a codon optimization approaches have been conducted and have obtained considerable amounts of recombinant protein, which is useful for academic studies and preclinical work [7,20].…”

Section: Discussionmentioning

confidence: 99%

“…and in contrast to LPS, are thought to be highly conserved. A large number of leptospiral OMPs have been characterized such as OmpL1 [15,16], LipL32 [17,18], LipL41 [19,20], LipL21 [7,21], Lig proteins [22], and Loa22 [23,24], etc., and evaluated the potential use of them in various immunodiagnostic assays either alone or in combination with other proteins. The OMPs play an important in maintaining the cell structure, and attachment to various extra cellular matrix (ECM) components, and are protective immunogens in animal models of the disease [25,26].…”

Section: Introductionmentioning

confidence: 99%

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Improvement the Expression and Purification of Loa22: A Lipoprotein with OmpA domain from Pathogenic Leptospira Serovars

Gharakhani

Ghasemi

Khaki

et al. 2022

Preprint

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Background Outer membrane proteins (OMPs) are the main surface antigens of the pathogenic Leptospires. One of the highly conserved outer membrane proteins expressed only by pathogenic Leptospires is Loa22. This study aims is to obtain the optimum conditions for high expression and purification of Loa22 recombinant (rLoa22) protein. Methods Based on evidence of phylogenetic studies, complete coding sequence of loa22 gene was optimized based on codon usage chart and sub-cloned into a pET32a (+) expression vector. BL21 (pLysS) was used as expression host for transformation. The recombinant clones were selected on ampicillin plates and subjected to PCR by using pET T7 primers and expression conditions optimized then by adjusting parameters such as culture media, induction time, temperature, and IPTG concentration. Results SDS-PAGE Analysis showed that the production of rLoa22 protein was at the highest level when post induction incubation, IPTG concentration, and duration of induction were 37ºC, 0.1M and 5h in 2xTY medium respectively. Due to the soluble nature of the protein, the purification of the rLoa22 protein under native conditions using Ni-NTA pull-down was optimum in one hour binding process at 37°C, five times washing process and elution buffer with a pH 7.4 and a 0.3 M imidazole concentration. Conclusion Based on the results of this study, optimizing the expression and purification process for over production of rLoa22 protein resulted in the large quantity of pure recombinant antigen that forms the basis for future investigation on the design of rapid diagnostic tests and more effective subunit vaccine candidates for leptospirosis.

show abstract

Improvement the expression and purification of Loa22: a lipoprotein with OmpA domain from pathogenic Leptospira serovars

Gharakhani

Ghasemi

Khaki

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Outer membrane proteins (OMPs) are the main surface antigens of the pathogenic Leptospires. One of the highly conserved outer membrane proteins expressed only by pathogenic Leptospires is Loa22. This study aims is to obtain the optimum conditions for high expression and purification of Loa22 recombinant (rLoa22) protein. Based on evidence of phylogenetic studies, complete coding sequence of loa22 gene was optimized based on codon usage chart and sub-cloned into a pET32a (+) expression vector. BL21 (pLysS) was used as expression host for transformation. The recombinant clones selected on ampicillin plates and subjected to PCR by using pET T7 primers and expression conditions optimized then by adjusting parameters such as culture media, induction time, temperature, and IPTG concentration. SDS-PAGE Analysis showed that the production of rLoa22 protein was at the highest level when post induction incubation, IPTG concentration, and duration of induction were 37ºC, 0.1 M and 5 h in 2xTY medium respectively. Due to the soluble nature of the protein, the purification of the rLoa22 protein under native conditions using Ni-NTA pull-down was optimum in one hour binding process at 37°C, five times washing process and elution buffer with a pH 7.4 and a 0.3 M imidazole concentration. Based on the results of this study, optimizing the expression and purification process for over production of rLoa22 protein resulted in the large quantity of pure recombinant antigen that forms the basis for future investigation on the design of rapid diagnostic tests and more effective subunit vaccine candidates for leptospirosis.

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Expression and purification of the Lipl41, a surface-exposed lipoprotein antigen of pathogenic Leptospira spp.

Cited by 3 publications

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Improvement the expression and purification of Loa22: a lipoprotein with OmpA domain from pathogenic Leptospira serovars

Improvement the expression and purification of Loa22: a lipoprotein with OmpA domain from pathogenic Leptospira serovars

Improvement the Expression and Purification of Loa22: A Lipoprotein with OmpA domain from Pathogenic Leptospira Serovars

Improvement the expression and purification of Loa22: a lipoprotein with OmpA domain from pathogenic Leptospira serovars

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