Expression and purification of the non-tagged LipL32 of pathogenic Leptospira

Hauk, Pricila; Carvalho, Enéas; Ho, Paulo Lee

doi:10.1590/s0100-879x2011007500025

Cited by 7 publications

(4 citation statements)

References 20 publications

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“…The genomic DNA of Leptospira interrogans serovar Canicola was used to amplify the lipL32 gene by PCR using published primers [13]. The reaction was carried out in a 25 μL reaction mixture containing 10X PCR buffer with MgCl 2 , 20 pmol of each primer, 0.2 mM dNTPs, 5 units of Vent polymerase, and 20 ng of template DNA.…”

Section: Polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

Serovar prevalence of Leptospira in semirural India and the development of an IgM-based indirect ELISA

Chandy

Lokeshwaran

Hemavathy

et al. 2017

J Infect Dev Ctries

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Introduction: Leptospirosis is a major public health problem in India. However, it has been underreported and under-diagnosed due to a lack of awareness of the disease, a functional surveillance system, and appropriate laboratory diagnostic facilities. Methodology: This multicenter study aimed to understand the Leptospira serovars causing leptospirosis in seven secondary-level hospitals in six states in India. Since early and accurate diagnosis of leptospirosis is one of the challenges faced by clinicians in India due to the poor specificity and sensitivity of commercially available diagnostic systems, an in-house indirect enzyme-linked immunosorbent assay (ELISA) was developed. Genomic DNA from L. interrogans serovar Canicola was used for polymerase chain reaction amplification, cloning, and expression of the lipL32 gene in E. coli to amplify, clone, and express the lipL32 gene. Results: Australis was the common serovar seen at all the study centers. Serovar Icterohaemorrhagiae was seen in samples from Tamil Nadu and Assam. In-house ELISA was standardized using the purified recombinant LipL32 polypeptide and was used to evaluate serum. Subsequently, acute serum samples from leptospirosis patients (n = 60) were screened. Compared to the gold standard, the microscopic agglutination test, sensitivity and specificity of the in-house ELISA was 95% and 90%, respectively. Conclusions: Understanding Leptospira serovars circulating in leptospirosis-endemic areas will help to formulate better vaccines. LipL32-based ELISA may serve as a valuable tool for early diagnosis of leptospirosis.

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Section: Polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

Serovar prevalence of Leptospira in semirural India and the development of an IgM-based indirect ELISA

Chandy

Lokeshwaran

Hemavathy

et al. 2017

J Infect Dev Ctries

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“…The soluble and insoluble fractions were isolated by centrifugation at 8400 ϫ g for 10 min. Purification of LipL32 mutants without a His tag fusion proceeded as previously described for wild-type LipL32 (20) followed by dialysis against 10 mM Tris-HCl (pH 8.0), 50 mM KCl. For the purification of LipL32 mutants with N-terminal His tag fusions, the soluble fraction of the cell lysate was applied onto a 5-ml Ni 2ϩ -charged chelating Sepharose bed (1 cm diameter column, GE Healthcare) equilibrated with 150 mM Tris-HCl (pH 8.0).…”

Section: Methodsmentioning

confidence: 99%

Calcium Binding to Leptospira Outer Membrane Antigen LipL32 Is Not Necessary for Its Interaction with Plasma Fibronectin, Collagen Type IV, and Plasminogen

Hauk

Barbosa

et al. 2012

Journal of Biological Chemistry

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LipL32 is the most abundant outer membrane protein from pathogenic Leptospira and has been shown to bind extracellular matrix (ECM) proteins as well as Ca 2؉ . Recent crystal structures have been obtained for the protein in the apo-and Ca 2؉ -bound forms. In this work, we produced three LipL32 mutants (D163-168A, Q67A, and S247A) and evaluated their ability to interact with Ca 2؉ and with ECM glycoproteins and human plasminogen. The D163-168A mutant modifies aspartate residues involved in Ca 2؉ binding, whereas the other two modify residues in a cavity on the other side of the protein structure. Loss of calcium binding in the D163-D168A mutant was confirmed using intrinsic tryptophan fluorescence, circular dichroism, and thermal denaturation whereas the Q67A and S247A mutants presented the same Ca 2؉ affinity as the wild-type protein. We then evaluated if Ca 2؉ binding to LipL32 would be crucial for its interaction with collagen type IV and plasma proteins fibronectin and plasminogen. Surprisingly, the wild-type protein and all three mutants, including the D163-168A variant, bound to these ECM proteins with very similar affinities, both in the presence and absence of Ca 2؉ ions. In conclusion, calcium binding to LipL32 may be important to stabilize the protein, but is not necessary to mediate interaction with host extracellular matrix proteins.

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“…The accurate result of serological test depends on the efficiency of the antigen also. The protein profile of outer membranes revealed the major band at approximately 32 kDa molecular weight (Haake et al, 2000;Abhinay et al, 2012), which is immunogenic (Hauk et al, 2011). The recombinant LipL32 (rLipL 32) protein is an optimal antigen for serodiagnosis of leptospirosis (Zhang et al, 2005) and ELISA based on this protein is a good diagnostic tool for leptospirosis (Dey et al, 2004), which is easier to perform, can accommodate a large number of samples and gives a less subjective result than MAT.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Two Rapid Field Level Diagnostic Tools for Acute Canine Leptospirosis in an Endemic Area

Ambily¹,

Mini²,

Joseph³

et al. 2019

Int.J.Curr.Microbiol.App.Sci

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Expression and purification of the non-tagged LipL32 of pathogenic Leptospira

Cited by 7 publications

References 20 publications

Serovar prevalence of Leptospira in semirural India and the development of an IgM-based indirect ELISA

Serovar prevalence of Leptospira in semirural India and the development of an IgM-based indirect ELISA

Calcium Binding to Leptospira Outer Membrane Antigen LipL32 Is Not Necessary for Its Interaction with Plasma Fibronectin, Collagen Type IV, and Plasminogen

Evaluation of Two Rapid Field Level Diagnostic Tools for Acute Canine Leptospirosis in an Endemic Area

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