Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the N-terminal carbohydrate-recognition domain of human galectin-4

Abstract

Galectin-4 is a tandem-repeat-type galectin that is expressed in the epithelium of the alimentary tract from the tongue to the large intestine. Additionally, strong expression of galectin-4 can also be induced in cancers in other tissues, including the breast and liver. In order to explore its potential as a target for anticancer drug design, elucidation of the structural basis of the carbohydrate-binding specificities of galectin-4 has been focused on. As an initial step, the N-terminal carbohydrate-recognition domain of human galectin-4 (hGal4-CRD-1) has been successfully crystallized using the vapour-diffusion technique, a complete data set has been collected to 2.2 Å resolution and the structure has been solved by the molecular-replacement technique. The crystals belonged to space group P6 1 22, with unit-cell parameters a = b = 71.25, c = 108.66 Å . The asymmetric unit contained one molecule of hGal4-CRD-1, with a V M value of 2.34 Å 3 Da À1 and a solvent content of 47.51%.

Recombinant expression, purification and preliminary biophysical and structural studies of C-terminal carbohydrate recognition domain from human galectin-4

“…Previously, hGal4-CRD1 domain was cloned, overexpressed, crystallized and its crystallographic structure determined [30]. In the present work, we move forward by developing reproducible protocols for heterologous expression, purification and crystallization of two constructs for hGal4-CRD2: the His 6 -tagged hGal4-CRD2 and tag-free hGal4-CRD2 forms.…”

“…2). The first construct, named pET28a-hGal4-CRD2 and based on pET28a expression vector, was inspired by the success of the expression and crystallization of the N-terminal His 6 -tagged hGal4-CRD1 domain [30]. However, possible His-tag interference with sugar binding was decisive in developing a new construct for expression of a tag-free protein.…”

Caracterização estrutural e avaliação de aspectos funcionais de galectinas humanas do grupo tandem-repeat

“…Os domínios hGal4-CRD1 e hGal4-CRD2 foram anteriormente clonados pelo nosso grupo no vetor pET-28a(+) (Novagen) (Zimbardi et al, 2010). As informações referentes à clonagem estão resumidas na tabela (tabela 3).…”

“…Os ensaios de cristalização da proteína nativa procederam conforme descrito (Zimbardi et al, 2010). Os cristais otimizados foram obtidos na presença de 0,1 M Tris-HCl (Sigma-Aldrich) pH 8,5, 16% (m/v) polietilenoglicol 8000 (Sigma-Aldrich) e 0,2 M acetato de cálcio hidratado (Sigma-Aldrich-Aldrich) a 21 °C.…”

“…Para a determinação das fases iniciais, foi utilizado como modelo de busca as coordenadas do modelo preliminar obtido a partir do uso do primeiro conjunto de dados obtido (Zimbardi et al, 2010). O refinamento da estrutura foi realizado utilizando o programa phenix.refine (Adams et al, 2010) seguido de vários ciclos de ajustes manuais do modelo feitos utilizando o programa COOT (Emsley et al, 2010), em que as posições atômicas do modelo inicial foram modificadas de forma a satisfazer a estereoquímica e o mapa de densidade eletrônica.…”