Process parameters optimization to produce the recombinant protein CFP10 for the diagnosis of tuberculosis.
Protein Expr Purif
; 154: 118-125, 2019 02.
Article
en En
| MEDLINE
| ID: mdl-30261310
ABSTRACT
The aim of this study was to evaluate the parameters that affect the production of the recombinant 10â¯kDa culture filtrate protein (CFP10), a promising reagent of high specificity for intradermoreaction and other antigen-based methods used in the diagnosis of tuberculosis. Conditions of Escherichia coli growth temperature, induction temperature and IPTG-inducer concentration were evaluated in shake flasks and dissolved O2 concentrations of 15 and 30% were evaluated in a bioreactor. The process parameters defined on small scale were growth temperature between 30 and 37⯰C, induction temperature of 26⯰C and IPTG concentration of 0.12â¯mM. The process conducted with 15% dissolved O2 presented a recombinant protein yield of 78.6â¯mgâ¯g-1 biomass and a proportion of recombinant protein (insoluble fraction) in relation to total insoluble protein of 72%, at the time of maximum productivity. The operation with 30% dissolved O2 resulted in lower recombinant protein yields of 62.9â¯mgâ¯g-1 biomass and 20% in relation to total insoluble protein, but in higher overall concentration in the culture broth (69.2â¯mgâ¯L-1versus 48.3â¯mgâ¯L-1). The protein identity was confirmed by mass spectrometry, showing high similarity to CFP10, 10â¯kDa of Mycobacterium tuberculosis H37Rv (score 95), and the purified antigen presented reactivity by the Western blotting assay.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Proteínas Bacterianas
/
Tuberculosis
/
Mycobacterium tuberculosis
/
Antígenos Bacterianos
Tipo de estudio:
Diagnostic_studies
Límite:
Humans
Idioma:
En
Revista:
Protein Expr Purif
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2019
Tipo del documento:
Article
País de afiliación:
Brasil