Overcoming problems to produce the recombinant protein LipL21 of Leptospira interrogans

Author(s)

Takahashi, Maria Beatriz ; Teixeira, Aline Florencio ; Nascimento, Ana Lucia Tabet Oller

• Document type: Journal article
• Source: BIOTECHNIQUES; v. N/A, p. 6-pg., 2023-04-17.

Abstract

The production of leptospiral recombinant proteins in the soluble form and in high yield from Escherichia coli is still a challenge. This work presents the cloning, expression and purification of the outer membrane protein of Leptospira interrogans, LipL21, which is considered an interesting target for vaccine and diagnostics development. The expression profile and yield of LipL21 was compared after cloning in the vectors pAE, pET28a and pET-SUMO, and it was observed that LipL21 was expressed in a low amount with pAE vector. By using the pET-28a vector, protein expression was increased, but the majority of the product was obtained as inclusion bodies. As a highlight, using a pET-SUMO vector was shown to overcome the problems of low expression and solubility of the lipoprotein LipL21.METHOD SUMMARYThe cloning, expression and purification of the outer membrane protein LipL21 of Leptospira interrogans was performed in the pAE, pET-28a and pET-SUMO vectors. By using the pET-SUMO vector, the addition of a SUMO tag at the N-terminal of the LipL21 protein improved the protein expression profile and yield. The target protein was obtained by a single-step purification using the following steps: affinity chromatography, protein cleavage, buffer exchange and protein elution. (AU)

• FAPESP's process: 14/50981-0 - Search for surface proteins among the genome sequences of Leptospira interrogans: functional and immunological characterization to understanding mechanisms involved in the bacterial pathogenesis
• Grantee: Ana Lucia Tabet Oller do Nascimento
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 19/17488-2 - Advancing the understanding of pathogenesis and virulence of Leptospira interrogans through proteomics, structural, mutagenesis and immunological analyses
• Grantee: Ana Lucia Tabet Oller do Nascimento
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 17/26223-7 - Evaluation of functional role of Leptospira interrogans surface proteins as endothelial and epithelial cells receptors of the host
• Grantee: Maria Beatriz Takahashi
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 16/11541-0 - Evaluation of the vaccine potential of antigenic fractions obtained in Leptospira interrogans serovar Copenhageni using hamsters as an animal model
• Grantee: Aline Florencio Teixeira
• Support Opportunities: Scholarships in Brazil - Post-Doctoral