Article

Efficient uniform isotope labeling of Abl kinase expressed in Baculovirus-infected insect cells.

Novartis Institutes for BioMedical Research, Basel, Switzerland.
Journal of Biomolecular NMR (Impact Factor: 2.85). 04/2005; 31(4):343-9. DOI: 10.1007/s10858-005-2451-3
Source: PubMed

ABSTRACT This report shows for the first time the efficient uniform isotope labeling of a recombinant protein expressed using Baculovirus-infected insect cells. The recent availability of suitable media for (15)N- and (13)C/(15)N-labeling in insect cells, the high expression of Abl kinase in these labeling media and a suitable labeling protocol made it possible to obtain a (1)H-(15)N-HSQC spectrum for the catalytic domain of Abl kinase of good quality and with label incorporation rates > 90%. The presented isotope labeling method should be applicable also to further proteins where successful expression is restricted to the Baculovirus expression system.

0 Bookmarks
 · 
50 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Protein production for structural and biophysical studies, functional assays, biomarkers, mechanistic studies in vitro and in vivo, but also for therapeutic applications in pharma, biotech and academia has evolved into a mature discipline in recent years. Due to the increased emphasis on biopharmaceuticals, the growing demand for proteins used for structural and biophysical studies, the impact of genomics technologies on the analysis of large sets of structurally diverse proteins, and the increasing complexity of disease targets, the interest in innovative approaches for the expression, purification and characterisation of recombinant proteins has steadily increased over the years. In this review, we summarise recent developments in the field of recombinant protein expression for research use in pharma, biotech and academia. We focus mostly on the latest developments for protein expression in the most widely used expression systems: Escherichia coli (E. coli), insect cell expression using the Baculovirus Expression Vector System (BEVS) and, finally, transient and stable expression of recombinant proteins in mammalian cells.
    Current Opinion in Structural Biology 05/2013; · 8.74 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The biochemical processes of living cells involve a numerous series of reactions that work with exceptional specificity and efficiency. The tight control of this intricate reaction network stems from the architecture of the proteins that drive the chemical reactions and mediate protein-protein interactions. Indeed, the structure of these proteins will determine both their function and interaction partners. A detailed understanding of the proximity and orientation of pivotal functional groups can reveal the molecular mechanistic basis for the activity of a protein. Together with X-ray crystallography and electron microscopy, NMR spectroscopy plays an important role in solving three-dimensional structures of proteins at atomic resolution. In the challenging field of membrane proteins, retinal-binding proteins are often employed as model systems and prototypes to develop biophysical techniques for the study of structural and functional mechanistic aspects. The recent determination of two 3D structures of seven-helical trans-membrane retinal proteins by solution-state NMR spectroscopy highlights the potential of solution NMR techniques in contributing to our understanding of membrane proteins. This review summarizes the multiple strategies available for expression of isotopically labeled membrane proteins. Different environments for mimicking lipid bilayers will be presented, along with the most important NMR methods and labeling schemes used to generate high-quality NMR spectra. The article concludes with an overview of types of conformational restraints used for generation of high-resolution structures of membrane proteins. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.
    Biochimica et Biophysica Acta 07/2013; · 4.66 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Because of the complexity, heterogeneity, and flexibility of the glycans, the structural analysis of glycoproteins has been eschewed until recently, with a few prominent exceptions. This aversion may have branded structural biologists as glycophobics. However, recent technological advancements in glycoprotein expression systems, employing genetically engineered production vehicles derived from mammalian, insect, yeast, and even bacterial cells, have yielded encouraging breakthroughs. The major advance is the active control of glycoform expression of target glycoproteins based on the genetic manipulation of glycan biogenetic pathways, which was previously overlooked, abolished, or considered unmanageable. Moreover, synthetic and/or chemoenzymatic approaches now enable the preparation of glycoproteins with uniform glycoforms designed in a tailored fashion.
    Current Opinion in Structural Biology 05/2014; 26C:44-53. · 8.74 Impact Factor