RESUMO
Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is a protein that binds and blocks the C5a receptor (C5aR) and formylated peptide receptor, thereby inhibiting the immune cell recruitment associated with inflammation. If CHIPS was less reactive with existing human antibodies, it would be a promising anti-inflammatory drug candidate. Therefore, we applied directed evolution and computational/rational design to the CHIPS gene in order to generate new CHIPS variants displaying lower interaction with human IgG, yet retaining biological function. The optimization was performed in four rounds: one round of random mutagenesis to add diversity into the CHIPS gene and three rounds of DNA recombination by Fragment INduced Diversity (FIND). Every round was screened by phage selection and/or ELISA for decreased interaction with human IgG and retained C5aR binding. The mean binding of human anti-CHIPS IgG decreased with every round of evolution. For further optimization, new amino acid substitutions were introduced by rational design, based on the mutations identified during directed evolution. Finally, seven CHIPS variants with low interaction with human IgG and retained C5aR blocking capacity could be identified.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Evolução Molecular Direcionada , Imunoglobulina G/imunologia , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Staphylococcus aureus/imunologia , Sequência de Aminoácidos , Proteínas de Bactérias/análise , Proteínas de Bactérias/farmacologia , Linhagem Celular , Desenho de Fármacos , Expressão Gênica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Staphylococcus aureus/genéticaRESUMO
The Chemotaxis Inhibitory Protein of Staphylococcus aureus (CHIPS) binds and blocks the C5a receptor (C5aR) and formyl-peptide receptor (FPR). This way, CHIPS is a potent inhibitor of the immune cell recruitment associated with inflammation. Truncation of the protein and the introduction of mutations, shifts the expression towards the insoluble fraction of Escherichia coli, whereas the wild-type protein can be solubly expressed. A protocol for expression and tag independent purification of biologically active CHIPS variants has been established to enable further characterization of an improved CHIPS variant, called ADC-1004. The CHIPS variants were purified by washing of E. coli inclusion bodies followed by refolding and gel filtration. New techniques were utilized to optimize the purification process. Expression in inclusion bodies was increased by the use of Ultra Yield flasks and optimal refolding conditions were determined by the use of the iFOLD Refolding System 2. The folding and biological activity of the purified proteins were analyzed by circular dichroism (CD) spectroscopy and flow cytometry, respectively, and compared to solubly produced CHIPS(31-113) and wild-type CHIPS(1-121). We show that the CHIPS variants produced in inclusion bodies can be refolded and purified to achieve equal biological activity as solubly produced CHIPS(31-113) and wild-type CHIPS(1-121). The truncation causes minor structural changes while purification from inclusion bodies or the soluble fraction does not further affect the structure.