Silkworm expression and sugar profiling of human immune cell surface receptor, KIR2DL1

Immune cell surface receptors are directly involved in human diseases, and thus represent major drug targets. However, it is generally difficult to Obtain Sufficient amounts of these receptors for biochemical and structural Studies because they often require post translational modifications, especially sugar modification Recently, we have established a bacmid expression system for the baculovirus BmNPV, which directly infects silkworms, an attractive host for the large-scale production of recombinant sugar-modified proteins. Here we produced the human immune cell surface receptor, killer cell Ig-like receptor 2DL1 (KIR2DL1), by Using the BmNPV bacmid expression system, in silkworms. By the direct injection of the bacmid DNA, the recombinant KIR2DL1 protein was efficiently expressed, secreted into body fluids, and purified by Ni2+ affinity column chromatography. We further optimized the expression conditions, and the final yield was 0.2 mg/larva. The sugar profiling revealed that the N-linked Sugars of the purified protein comprised very few components, two paucimannose-type oligosaccharides, Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc and Man alpha 1-6Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc. This revealed that the protein product was Much more homogeneous than the complex-sugar type Product obtained by mammalian cell expression. The Surface plasmon resonance analysis demonstrated that the purified KIR2DL1 protein exhibited specific binding to the HLA-Cw4 ligand. Moreover, the CD spectrum showed the proper secondary Structure. These results clearly Suggested that the silkworm expression system is quite useful for the expression of cell surface receptors that require posttranslational modifications, as well as for then-structural and binding studies, due to the relatively homogeneous N-linked sugar modifications. (C) 2009 Elsevier Inc. All rights reserved.