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Vol. 21. Issue 2.
Pages 82-89 (July - December 2020)
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Vol. 21. Issue 2.
Pages 82-89 (July - December 2020)
Original article
Purification of the human papillomavirus 16 L1 protein from E. coli SHuffle® T7
Purificación de la proteína L1 del virus del papiloma humano tipo 16 a partir E.coli SHuffle® T7
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S.M. Brito Molinaa,
Corresponding author
susanabrito@gmx.es

Corresponding author.
, Y. Serrano Riveroa, A. Falero Morejóna, E. Pimienta Rodrígueza, S. Rodríguez Salgueiroa, O. Ancheta Nieblab, K. Marro Domíngueza
a Centro Nacional de Investigaciones Científicas, La Habana, Cuba
b Universidad Latinoamericana de Ciencias Médicas, La Habana, Cuba
Highlights

  • HPV-16 L1-His protein represented ∼ 12% of total proteins.

  • It was purified to a purity of ∼ 90% by means of Ni2+ ion affinity chromatography.

  • 9 mg de pentamers of cultured L1-His/L was obtained after renaturalisation.

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Abstract
Objective

To purify the Human Papillomavirus 16 (HPV-16) L1 protein encoded by a cloned L1 gene from a sample of cervical cancer from a Cuban patient and fused to a histidine tag at its carboxyl end (L1-His) from E. coli SHuffle® T7.

Methods

The strain E. coli SHuffle® T7 with the plasmid pETHPV16L1myc-His was used to obtain the HPV-16 L1-His protein, grown under autoinduction conditions. L1-His protein was purified by Ni2+ ion-chelated affinity chromatography (Ni2+-IMAC), after extraction of the inclusion bodies (IB) with 8 M urea and subsequent refolding by an inverse dilution method. The molecular size of the refolded HPV-16 L1-His protein was analyzed by native polyacrilamide gel electrophoresis and molecular exclusion chromatography.

Results

The HPV-16 L1-His protein accumulated in IB in E. coli SHuffle® T7 and represented ∼12% of total proteins. After its extraction with 8 M urea from IB was purified by Ni2+-IMAC, with an ∼90% purity and an ∼40% yield. Renaturation of purified L1-His allowed obtaining ∼9 mg of pentamers/L of culture, with a final recovery of ∼62%.

Conclusions

For the first time it was described the purification of pentamers of HPV-16 L1-His protein encoded by a cloned L1 gene from a Cuban patient from E. coli SHuffle® T7´s IB. The developed strategy could be an alternative for obtaining L1-His protein capsomers for developing a vaccine candidate against HPV-16.’

Keywords:
HPV
Escherichia coli
L1 protein
Purification
Inclusion body
Resumen
Objetivo

Purificar la proteína L1 del Virus del Papiloma Humano 16 (VPH-16) codificada por un gen L1 clonado de una muestra de cáncer cervical de una paciente cubana y fusionada por su extremo carboxilo a una cola de histidinas (L1-His), a partir de Escherichia coli SHuffle® T7.

Métodos

La cepa E. coli SHuffle® T7 transformada con el plásmido pETHPV16L1myc-His se empleó para producir la proteína L1-His del VPH-16 cultivada en condiciones de autoinducción. La proteína L1-His se purificó mediante cromatografía de afinidad de quelatos de iones Ni2+ (IMAC-Ni2+), tras extracción de los cuerpos de inclusión (CI) con urea 8 M y posterior renaturalización por dilución inversa. La talla molecular de la proteína L1-His renaturalizada se evaluó mediante electroforesis nativa y cromatografía de exclusión molecular.

Resultados

La proteína L1-His del VPH-16 se acumuló en CI en E. coli SHuffle® T7, representando ∼12% de las proteínas totales. Se purificó mediante IMAC-Ni2+ en condiciones desnaturalizantes, con una pureza de ∼90% y un rendimiento de ∼40%. La renaturalización de la L1-His purificada permitió obtener ∼9 mg de pentámeros/L de cultivo, con un rendimiento final del proceso de ∼62%.

Conclusiones

En este trabajo se describió por primera vez la purificación de pentámeros de la proteína L1-His del VPH-16, codificada por un gen L1 clonado de una muestra de cáncer cervical de una paciente cubana, a partir de los CI de E. coli SHuffle® T7. La estrategia desarrollada podría ser una alternativa para la obtención de capsómeros de L1-His, para desarrollar un candidato vacunal contra el VPH-16.

Palabras clave:
VPH
Escherichia coli
Proteína L1
Purificación
Cuerpos de inclusión

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