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2019, Number 2

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Rev Cubana Med Trop 2019; 71 (2)

Cloning and expression in Escherichia coli of the full-length and deletion variants of a human papillomavirus 18 L1 gene isolated from a Cuban patient

Pimienta E, Rodríguez S, Fando R, Serrano Y, Ortega D, Palenzuela A, Marrero K

Language: English
References: 55
Page: 1-25
PDF size: 589.26 Kb.


ABSTRACT

Introduction: Human papillomavirus (HPV) vaccines are based on the L1 major capsid protein.
Objectives: To clone the HPV-18 L1 gene from a Cuban female HPV-18-infected patient and to express the full-length and deletion variants of the cloned HPV-18 L1 gene in Escherichia coli.
Methods: The full-length HPV-18 L1 gene was PCR-amplified from total DNA isolated from a Cuban patient, cloned and finally subcloned into the E. coli expression vector pET26b. Three deletion mutants were constructed, which encode truncated proteins lacking 30 amino acids at the C-terminus in combination with 5, 6 or none deleted residue at the N-terminus. Production of L1 proteins in E. coli BL21(DE3) and E. coli SHuffle T7 was assessed by SDS-PAGE and Western blotting.
Results: The cloned HPV-18 L1 gene was 99.9 % similar to the African variant EF202152 and probably shares a common origin with the B lineage of genotype 18. The three truncated variants of HPV-18 L1 were produced at higher levels than the full-length HPV-18 L1 protein, attaining higher levels in E. coli BL21(DE3) and higher solubility in E. coli SHuffle. The C-terminus-only truncated variant, L1ΔC30, was produced at similar levels to the HPV-18 L1s truncated at both termini. E. coli SHuffle produced about three times more amounts of L1ΔC30 when grown under autoinduction conditions with respect to conventional induction and thus, amounts were comparable to those obtained in E. coli BL21(DE3) under conventional induction.
Conclusions: Truncation of thirty amino acid residues at the carboxy-terminus of the HPV-18 L1 made a major contribution to the production and solubility of this wild-type protein in E. coli. This is the first report about soluble production of HPV-18 L1 protein in an E. coli SHuffle strain. However, higher amounts of L1 are needed to scale-up its production for developing an HPV vaccine candidate.


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