Overcoming problems to produce the recombinant protein LipL21 of Leptospira interrogans

Autor(es)

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

• Tipo de documento: Artigo Científico
• Fonte: BIOTECHNIQUES; v. N/A, p. 6-pg., 2023-04-17.

Resumo

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)

• Processo FAPESP: 14/50981-0 - Busca de proteínas de superfície nas sequências do genoma da Leptospira interrogans: caracterização funcional e imunológica para o entendimento de mecanismos envolvidos na patogênese de bactéria
• Beneficiário: Ana Lucia Tabet Oller do Nascimento
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 19/17488-2 - Avançando no entendimento da patogenicidade e virulência da Leptospira interrogans através de análises proteômicas, estruturais, mutagênicas e imunológicas
• Beneficiário: Ana Lucia Tabet Oller do Nascimento
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 17/26223-7 - Avaliação do papel funcional de proteínas de superfície de Leptospira interrogans como receptores de células endoteliais e epiteliais do hospedeiro
• Beneficiário: Maria Beatriz Takahashi
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto
• Processo FAPESP: 16/11541-0 - Avaliação do potencial vacinal de frações antigênicas obtidas em Leptospira interrogans serovar Copenhageni utilizando hamster como modelo animal
• Beneficiário: Aline Florencio Teixeira
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado