Recombinant erythropoietin (rhEPO) is often used as “doping agent” by athletes in endurance sports to increase blood oxygen capacity. Some strategies improve the pharmacological properties of erythropoietin (EPO) through the genetic and chemical modification of the native EPO protein. The EPO-Fcs are fusion proteins composed of monomeric or dimeric recombinant EPO and the dimeric Fc region of human IgG molecules. The Fc region includes the hinge region and the CH2 and CH3 domains. Recombinant human EPOs (rhEPO) fused to the IgG Fc domain demonstrate a prolonged half-life and enhanced erythropoietic activity in vivo compared with native or rhEPO.
Drug-testing agencies will need to obtain primary structure information and develop a reliable analytical method for the determination of EPO-Fc abuse in sport. The possibility of EPO-Fc detection using nanohigh-performance liquid chromatography−tandem mass spectrometry (HPLC−MS/MS) was already demonstrated (1). However, the prototyping peptides derived from EPO and IgG are not selective enough because both free proteins are naturally presented in human serum. In a recent publication, researchers describe the effort to identify peptides covering unknown fusion breakpoints (later referred to as “spacer” peptides; 2). The identification of “spacer” peptides will allow the confirmation of the presence of exogenous EPO-Fc in human biological fluids.
A bottom-up approach and the intact molecular weight measurement of deglycosylated protein and its IdeS proteolytic fractions was used to determine the amino acid sequence of EPO-Fc. Using multiple proteases, peptides covering unknown fusion breakpoints (spacer peptides) were identified.
Results indicated that “spacer peptides” could be used in the determination of EPO-Fc fusion proteins in biological samples using common LC−tandem MS methods.
- Reichel, C. et al. (2012) Detection of EPO-Fc fusion protein in human blood: screening and confirmation protocols for sports drug testing.
Drug Test. Anal. 4, 818−29.
- Mesonzhnik, N. et al. (2017) Characterization and Detection of Erythropoietin Fc Fusion Proteins Using Liquid Chromatography−Mass Spectrometry.
J. of Proteome Res. 17, 689-97.
Endo H (Endo-ß-N-acetylglucosaminidase H) is a 29,000 dalton protein with optimal activity between pH 5 and 6. In contrast to PNGase F, which cleaves all N-linked glycans at the site of attachment to Asparagine (Asn), (with the exception of those with fucose attached to the core GlcNac moieties), Endo H hydrolyses the bond connecting the two GlcNac groups that comprise the chitobiose core (see Figure 1.). In addition, Endo H cleaves high mannose and hybrid glycans, whereas complex glycans (those with more than 4 different sugar types per glycan chain, including the GlcNac groups) are resistant to hydrolysis.
The unique specificty of Endo H and PNGase F can be used to monitor protein trafficking. Basic N-Glycosylation occurs in the endoplasmic reticulum. Proteins in this stage are sensitive to Endo H digestion. If proteins have entered the Golgi body where additional modifications occur to the glycan, they are resistant to Endo H digestion.
The following references illustrate this application:
- Taner, S. et al. (2011) Interactions of NK cell receptor KIR3DL1*004 with chaperones and conformation-specific antibody reveal a functional folded state as well as predominant intracellular retention. J. Immunol. 186, 62–72
- Beyer, S. et al. (2011) KT5823 differentially modulates sodium iodide symporter expression, activity, and glycosylation between thyroid and breast cancer cells. Endo. 152, 782–92.
- Chen, Z. et al. (2011) CEACAM1 dampens antitumor immunity by down-regulating NKG2D ligand expression on tumor cells. J. Exper. Med. 208, 2633–40.
- Iavarone, C. et al. (2011) A point mutation in the amino terminus of TLR7 abolishes signaling without affecting ligand binding. J. Immunol. 186, 4213–22.
- Guo, Y. et al. (2011) Herpes simplex virus encephalitis in a patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective immunity. J. Exper. Med. 208, 2083–98.
Many proteins are expressed as fusion partners with affinity tags, such as HaloTag, glutathione-S-transferase (GST) or maltose binding protein (MBP), to selectively bind the proteins using affinity purification resins. While such resins yield high-purity protein quickly, the large affinity tags are undesirable for some downstream applications. Most expression vectors are designed with a specific protein cleavage site between the two fusion partners to remove the affinity tag after purification. ProTEV Protease recognizes a rare amino acid sequence, EXXYXQ, where X is any amino acid and cleavage occurs after the glutamine residue. TEV protease will cleave proteins with 19 of the 20 amino acids present after the glutamine residue; the exception is proline . Continue reading
Two of the most frequent applications that use cell-free expression are the characterization of protein:protein interactions and the characterization of protein:nucleic acid interactions. Due to the convenience of expressing functional protein in few hours, cell-free expression is also a viable alternative to cell-based expression for other applications. Recent examples include: Continue reading