Effect of pressure on refolding of recombinant pentameric cholera toxin B.
The production of recombinant proteins is an essential tool for the expansion of modern biological research and biotechnology. The expression of heterologous proteins in Escherichia coli often results in an incomplete folding process that leads to the accumulation of inclusion bodies (IB), aggregates that hold a certain degree of native-like secondary structure. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, leading to dissociation of aggregates under non-denaturing conditions and is therefore a useful tool to solubilize proteins for posterior refolding. Cholera toxin (CT) is composed of a non-toxic pentamer of B subunits (CTB), a useful adjuvant in vaccines, and a toxic subunit A (CTA). We studied the process of refolding of CTB using HHP. HHP was shown to be effective for dissociation of CTB monomers from IB. Posterior incubation at atmospheric pressure of concentrated CTB (1mg/ml) is necessary for the association of the monomers. Pentameric CTB was obtained when suspensions of CTB IB were compressed at 2.4kbar for 16h in the presence of Tween 20 and incubated at 1bar for 120h. Soluble and biologically active pentameric CTB was obtained, with a yield of 213mg CTB/liter of culture. The experience gained in this study can be important to improve the refolding of proteins with quaternary structure.
Instituto de Pesquisas Energéticas e Nucleares - IPEN - CNEN/SP, Centro de Biotecnologia, São Paulo, Brazil.
D
D
Rodrigues
Universidade de São Paulo, Departamento de Microbiologia do Instituto de Ciências Biomédicas, São Paulo, Brazil.
L E
LE
Farinha-Arcieri
Universidade de São Paulo, Departamento de Microbiologia do Instituto de Ciências Biomédicas, São Paulo, Brazil.
A M
AM
Ventura
Instituto de Pesquisas Energéticas e Nucleares - IPEN - CNEN/SP, Centro de Biotecnologia, São Paulo, Brazil.
R M
RM
Chura-Chambi
Instituto de Pesquisas Energéticas e Nucleares - IPEN - CNEN/SP, Centro de Biotecnologia, São Paulo, Brazil.
N V
NV
Malavasi
Instituto de Pesquisas Energéticas e Nucleares - IPEN - CNEN/SP, Centro de Biotecnologia, São Paulo, Brazil.
L S
LS
Lemke
Instituto de Pesquisas Energéticas e Nucleares - IPEN - CNEN/SP, Centro de Biotecnologia, São Paulo, Brazil.
J S
JS
Guimarães
Instituto Butantan, Centro de Biotecnologia, São Paulo, Brazil.
P L
PL
Ho
Instituto de Pesquisas Energéticas e Nucleares - IPEN - CNEN/SP, Centro de Biotecnologia, São Paulo, Brazil. Electronic address: lmorganti@ipen.br.
L
L
Morganti
D002772:Cholera Toxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D006874:Hydrostatic Pressure / Q000009:adverse effects; D008958:Models, Molecular; D011487:Protein Conformation; D055503:Protein Multimerization; D058849:Protein Refolding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014734:Vibrio cholerae / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D002772:Cholera Toxin
Aggregates; Cholera toxin B; High hydrostatic pressure; Inclusion bodies; Refolding