Expression, purification and characterization of galectin-1 in Escherichia coli.

As a member of beta-galactoside-binding proteins family, Galectin-1 (Gal-1) contains a single carbohydrate recognition domain, by means of which it can bind glycans both as a monomer and as a homodimer. Gal-1 is implicated in modulating cell-cell and cell-matrix interactions and may act as an autocrine negative growth factor that regulates cell proliferation. Besides, it can also suppress TH1 and TH17 cells by regulating dendritic cell differentiation or suppress inflammation via IL-10 and IL-27. In the present study, Gal-1 monomer and concatemer (Gal-1②), which can resemble Gal-1 homodimer, were expressed in Escherichia coli and their bioactivities were analyzed. The results of this indicate that both Gal-1 and Gal-1② were expressed in E. coli in soluble forms with a purity of over 95% after purifying with ion-exchange chromatography. Clearly, both Gal-1 and Gal-1② can effectively promote erythrocyte agglutination in hemagglutinating activity assays and inhibit Jurkat cell proliferation in MTT assays. All these data demonstrate that bacterially-expressed Gal-1 and Gal-1② have activities similar to those of wild type human Gal-1 whereas the bioactivity of concatemer Gal-1② was stronger than those of the bacterially-expressed and wild type human Gal.

The Escherichia coli-derived thymosin ß4 concatemer promotes cell proliferation and healing wound in mice.

Thymosin ß4 (Tß4) is one of the most promising thymosins for future clinical applications, and it is anticipated that commercial demand for Tß4 will increase. In order to develop a new approach to produce recombinant Tß4, a 168 bp DNA (termed Tß4) was designed based on the Tß4 protein sequence and used to express a 4 × Tß 4 concatemer (four tandem copies of Tß4, termed 4 × Tß4) together with a histidine tag (6 × His) in E. coli (strain BL21). SDS-PAGE and western blot analysis were used to confirm that a recombinant 4 × Tß4 protein of the expected size (30.87 kDa) was produced following the induction of the bacterial cultures with isopropyl ß-D-thiogalactoside (IPTG). The E. coli-derived 4 × Tß4 was purified by Ni-NTA resin, and its activities were examined with regard to both stimulating proliferation of the mice spleen cells in vitro and in vivo wound healing. The results demonstrate that these activities of the E. coli-derived recombinant 4 × Tß4 were similar or even better than existing commercially obtained Tß4. This production strategy therefore represents a potentially valuable approach for future commercial production of recombinant Tß4.

Tumor associated release of interleukin-10 alters the prolactin receptor and down-regulates prolactin responsiveness of immature cortical thymocytes.

The soluble factors produced either by Ehrlich's ascites carcinoma (EAC) or thymic adherent cells (TAC) of tumor-bearing mice comprising of CD11b(+) and CD11c(+) antigen-presenting cells caused a sharp decrease in prolactin (PRL)-induced ConA-mediated effect on survival of PNA(+) thymocytes. Similar suppression of PRL-induced effect was observed when the cells were cocultured with TAC of EAC-bearing mice. Anti-IL-10 antibody could reverse the PRL inability to induce ConA-mediated effect on PNA(+) thymocyte survival, indicating the presence of IL-10 in EAC culture supernatant (EAC sup) and thymic microenvironment. IL-10 could block PRL-induced proliferation of PNA(+) thymocytes without affecting spontaneous apoptosis. IL-10 altered the expression of the long-form (LF) of PRL-R and reduced the PRL binding of the cells, suggesting down-regulation of the PRL effect on PNA(+) thymocyte by the cytokine. Induction of tumor, which was found to increase the IL-10 secretion by TAC, also modified the PRL-R (LF) to PRL-R (SF). Since PRL plays a role in survival, proliferation and differentiation of lymphoid progenitor cells, the tuning of PRL action by IL-10 may be a possible mechanism of depletion of immature cortical thymocytes and thymic atrophy in tumor-bearing mice.