60ª Reunião Anual da SBPC

INTRODUÇÃO:

Ki-1/57, the 57-kDa human protein antigen recognized by the CD30 antibody Ki-1, is a cytoplasmic and nuclear protein that is phosphorylated on serine and threonine residues. The human protein Ki-1/57 is phylogenetically ancient, because we can find orthologues in both vertebrates and invertebrates, including Drosophila melanogaster, Caenorhabditis elegans, and Aedes aegypti. However, little is known about the function and structure of Ki-1/57. The low protein expression yield of full length protein 6xHis-Ki-1/57 so far did not allow us to perform successful crystallization experiments.

METODOLOGIA:

Our strategy here was to align the orthologues of Ki-1/57 which were found in diverse species of mammals, to identify the most conserved regions for cloning, expression and purifying these fragments, in an attempt to obtain pure protein in greater concentration. These fragments belong to regions well conserved in different organisms, and therefore it may be that they have a greater tendency to have a defined “domain” structure. To test this hypothesis we will perform structural assays with these two fragments of the protein Ki-1/57. The other technique performed in this work is the "Reverse Two Hybrid", which will make it possible to study detailed aspects of interactions between proteins. Initially, we construct cDNAs mutants of the Ki-1/57 protein through a PCR reaction in the presence of high concentrations of Magnesium. These random mutants of Ki-1/57 were then tested for their ability to interact with the protein RACK-1. The mutants that do not interact with RACK1 were identified by the b-galactosidase assay (white colonies) and their plasmids were isolated, sequenced and analyzed to identify which residues of amino acids have been mutated that were essential for interaction.

RESULTADOS:

Bioinformatics studies served to define the conserved regions between ortologues of Ki-1/57 of different species. The conserved regions of Ki-1/57 were expressed in soluble form in pET28a-TEV and pET28a-GST-TEV. The next step is to optimize the purification conditions, attempting to reduce the protein degradation and try to crystallize the purified protein. After testing several conditions to standardize the amount of mutations that was produced by PCR in C3 fragment of Ki-1/57 the condition that was more promising is between 1 and 2 mM MnCl. Now we are producing the mutants on a large scale to analyze the mutations and try to identify the essential amino acids for interaction with the protein RACK-1.

CONCLUSÕES:

The bioinformatics analysis of the ortologues of Ki-1/57 enabled us to find conserved regions, which were cloned into vectors for expression. The tests of expression show the proteins in the soluble fraction, but after purification are identified 2 bands in the gel probably because of degradation. It will be necessary to optimize the conditions for purification and reduce the protein degradation that can be observed in the polyacrylamide gel. Tests for standardization of random mutations in PCR (polymerase chain reaction) were performed successfully. We are currently sequencing in order to identifying those mutants of the protein Ki-1/57 that have lost the ability to interact with the protein RACK-1, and that are essential for this interaction.

Instituição de fomento: FAPESP

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E-mail para contato: kali_bio@yahoo.com.br