[Show abstract][Hide abstract] ABSTRACT: Whereas bacterial expression systems are widely used for production of uniformly or selectively (15)N-labeled proteins the usage of the baculovirus expression system for labeling is limited to very few examples in the literature. Here we present the complete formulations of the two insect media, IML406 and 455, for the high-yield production of selectively (15)N-labeled proteins in insect cells. The quantities of (15)N-amino acids utilized in the production of labeled GST were similar in the case of bacterial and viral expression. For the most studied amino acids essential for insect cells the (15)N-HSQC spectra, recorded with GST labeled in insect cells, showed no cross labeling and provided therefore spectra of better quality compared to NMR spectra of GST expressed in E. coli. Also in the case of amino acids not essential for Sf9 cells we were able to label a defined number of amino acid species. Therefore the selective labeling using the baculovirus expression vector system represents a complement or even an alternative to the bacterial expression system. Based on these findings we can provide a first simple overview of the network of the amino acid metabolism in E. coli and insect cells focused on nitrogen. For some amino acids the expression of labeled proteins in insect cells can replace the cell-free protein expression.
Full-text · Article · May 2003 · Journal of Biomolecular NMR
[Show abstract][Hide abstract] ABSTRACT: Melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma, as enhanced values diagnose metastatic melanoma stages III and IV. Here we show that the recombinant human MIA adopts an SH3 domain-like fold in solution, with two perpendicular, antiparallel, three- and five-stranded β-sheets. In contrast to known structures with the SH3 domain fold, MIA is a single-domain protein, and contains an additional antiparallel β-sheet and two disulfide bonds. MIA is also the first extracellular protein found to have the SH3 domain-like fold. Furthermore, we show that MIA interacts with fibronectin and that the peptide ligands identified for MIA exhibit a matching sequence to type III human fibronectin repeats, especially to FN14, which is close to an integrin α4β1 binding site. The present study, therefore, may explain the role of MIA in metastasis in vivo, and supports a model in which the binding of human MIA to type III repeats of fibronectin competes with integrin binding, thus detaching cells from the extracellular matrix.
[Show abstract][Hide abstract] ABSTRACT: The kinase CDK6 was expressed as different fusion proteins using the baculovirus expression vector system in high yields and afterwards purified. The fusion proteins were obtained in high purity and possessed a high stability. The cleavage of GST-CDK6 with thrombin resulted in homogenous CDK6 with reduced stability. For production of selectively with <sup>15</sup>N-amino acids labelled proteins in insect cells (Sf9) the media IML406 and IML455 were developed. In the most cases selective labelling of GST with these media in Sf9 yielded in better NMR spectra compared to the results in E. coli. The reason is the limited possibility of cross-labelling in Sf9. This fact enables the labelling of a higher number of amino acids with high yields in Sf9 compared to E. coli. Selective labelling in Sf9 can be an alternative to in vitro translation.