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.
"Thanks to the reduced metabolism of insect cells, for 15 N labeling only the pure amino acids and the yeast extract need to be taken into account, whereas for 13 C labeling also the carbohydrates need to be considered. There are protocols for uniform labeling, where the important nitrogen and/or carbon sources are replaced by labeled ones (Strauss et al. 2005; Walton et al. 2006), but this remains prohibitively expensive for most laboratories and is not the subject of this work. Nevertheless, in many instances uniform labeling is not necessary and the experimental question can be solved by only labeling selected sites in a protein. "
[Show abstract][Hide abstract] ABSTRACT: An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract, similar protein amounts as with full media are obtained, without compromising on isotope incorporation. In single and dual amino acid labeling experiments incorporation ratios are consistently ≥90% for all amino acids tested. This enables NMR studies of eukaryotic proteins and their interactions even for proteins with low expression levels. We show applications with human kinases, where protein-ligand interactions are characterized by 2D [(15)N, (1)H]- and [(13)C, (1)H]-HSQC spectra.
"In the past decade, a variety of such molecules have been produced in animal cell lines using recombinant technology (e.g. Bosman et al. 2003; Strauss et al. 2005; Ratnala 2006; Werner et al. 2008), but stable-isotope labeling in such cell lines is still extremely expensive. While many proteins can be functionally produced in prokaryotic systems like Escherichia coli, allowing affordable stable-isotope labeling (Ross et al. 2004), most eukaryotic membrane proteins, including most drug targets, cannot be produced in such systems in sufficient functional quantity or purity. "
[Show abstract][Hide abstract] ABSTRACT: Uniform stable-isotope labeling of mammalian cells is achieved via a novel formulation of a serum-free cell culture medium that is based on stable-isotope-labeled autolysates and lipid extracts of various microbiological origin. Yeast autolysates allow complete replacement of individual amino acids and organic acids in a chemically defined medium (DMEM/F12), enabling a cost-effective formulation of a stable-isotope-labeled culture medium for mammalian cells. In addition, biomass-derived hydrolysates, autolysates, and lipid extracts of various classes of algae were explored as cell culture components, both separately and in combination with yeast autolysates. Optimal autolysate concentrations were established. Such novel medium formulations were tested on mammalian cell lines, often used for recombinant protein production, i.e., Chinese hamster ovary (CHO) and human embryonic kidney (HEK 293). Special attention was paid to the adaptation of these mammalian cell lines to serum-free media. Formulation of the novel proprietary cell culture medium PLIm, based on yeastolates instead of individual amino acids and organic acids, allows a four- to eightfold cost reduction for (15)N and (13)C,(15)N stable-isotope-labeling, respectively, in CHO cells and a three- to sixfold cost reduction in HEK 293 cells. A high level of stable-isotope enrichment of mammalian cells (>90%) was achieved within four passages by complete replacement of carbon and nitrogen sources in the medium with their stable-isotope-labeled analogs. These conditions can be used to more cost-effectively produce labeled recombinant proteins in mammalian cells.
[Show abstract][Hide abstract] ABSTRACT: Over half of the proteins found in nature are anticipated to be glycosylated, yet less than 2% of the protein structures in the protein data bank are glycoproteins. Clearly, we are behind in our structural understanding of glycoproteins. One reason for the lack of structural information for glycoproteins is due to their oligosaccharide heterogeneity. The two main methods for determining detailed three-dimensional protein structures are X-ray crystallography and nuclear magnetic resonance. Oligosaccharide heterogeneity hinders crystallization for X-ray crystallography analysis and complicates resonance assignment of nuclear magnetic resonance data. Another challenge is expressing high enough yields from eukaryotic cells for X-ray crystallography and nuclear magnetic resonance analysis. Additionally, unlike bacteria, eukaryotic cell lines typically cannot synthesize amino acids from glucose and ammonium chloride. Thus, eukaryotic media must contain amino acids for cell growth and protein production. Structural analysis of glycoproteins by nuclear magnetic resonance requires that the amino acids are labeled with 13C- and 15N and that the oligosaccharides are labeled with 13C. Media containing 13C- and 15N-labeled amino acids is extremely expensive. This dissertation explains methods that address each of these challenges utilizing the glycoprotein Thy-1. An economic approach for labeling recombinant protein expressed from insect cells for NMR is explained. A method for labeling oligosaccharides on glycoproteins expressed from mammalian cells is also explained. Changes in previously established expression and purification protocols for Thy-1 derived from Lec1 cells, which produce relatively homogeneous glycoforms, have increased yields to levels suitable for nuclear magnetic resonance studies. In addition, the biological relevancy of recombinant Thy-1, which is expressed with truncated oligosaccharides and without it’s glycosylphosphatidylinositol anchor, was confirmed by receptor-binding analysis.
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.