The modular architecture of leukocyte cell‐surface receptors

Abstract

Cells of the immune system have a large number of protein receptors on their surfaces, with a wide range of binding functions. They are, however, constructed from a limited set of protein structural units, which are recognisable at the sequence level. The 3D structure of many of these domains, or modules, is now known. These modular units and their structures are reviewed here. The ways in which they are assembled into multidomain receptor chains and oligomeric complexes of receptors are also discussed.

Dictyostelium discoideum as Expression Host: Isotopic Labeling of a Recombinant Glycoprotein for NMR Studies

“…1). Secretion of recombinant protein was maintained until a cell count of 10 7 /ml, at which point growth cultures were processed to recover secreted [U- 13 C, 15 N]-PsA. Figure 1A shows the detection of secreted recombinant product throughout cell growth by SDS-PAGE, indicating the significant quantity of recombinant product available by day 7.…”

“…All cells were then removed from the growth medium by centrifugation (7000g, 20 min). The supernatant, containing secreted [U- 13 C, 15 N]-PsA, was filtered (0.45-m membrane) and concentrated to 10 ml (i.e., to a concentration of 4 mg/ml) in a stirred cell unit over a 10-kDa-cutoff membrane. Further yield of isotopically labeled protein product could be obtained by resuspending the D. discoideum cell pellet in Mes buffer (20 mM, pH 6.5) and shaking overnight at 21°C.…”

“…The former system was demonstrated to yield good levels of protein expression, but many steps of purification are required to isolate the recombinant protein from extracellular carbohydrate (8). Labeling using the mammalian CHO system has not been pursued in recent years, due to the expense of preparing appropriate culture conditions incorporating 13 C and 15 N labels. The ability of D. discoideum to feed on E. coli cells preenriched with the appropriate isotopic labels therefore opens a new avenue for the low-cost production in a eukaryotic host of protein samples for structure elucidation.…”

“…The primary structure of wild-type PsA has been well characterized (15,16). The protein comprises (i) an N-terminal globular domain of 90 residues, (ii) a heavily O-glycosylated linker consisting of variable numbers (in different isolates) of a Pro-Thr-Val-Thr tandem repeat, and (iii) a glycosyl phosphatidyl inositol (GPI) anchor, which is covalently bound to the Cterminal glycine and anchors the glycoprotein to the cell membrane.…”

“…The high-resolution protein structures being obtained today from modern triple-resonance NMR techniques rely on the generation of milligram quantities of protein labeled with 13 C, 15 N, and 2 H isotopes (5). Escherichia coli continues to be the most common system used for the production of these protein samples.…”