The tyrosine kinase Met, the product of the c-met proto-oncogene and the receptor for hepatocyte growth factor/scatter factor (HGF/SF), mediates signals critical for cell survival and migration. The human pathogen Listeria monocytogenes exploits Met signaling for invasion of host cells via its surface protein InlB. We present the crystal structure of the complex between a large fragment of the human Met ectodomain and the Met-binding domain of InlB. The concave face of the InlB leucine-rich repeat region interacts tightly with the first immunoglobulin-like domain of the Met stalk, a domain which does not bind HGF/SF. A second contact between InlB and the Met Sema domain locks the otherwise flexible receptor in a rigid, signaling competent conformation. Full Met activation requires the additional C-terminal domains of InlB which induce heparin-mediated receptor clustering and potent signaling. Thus, although it elicits a similar cellular response, InlB is not a structural mimic of HGF/SF.

BackgroundThe demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility.ResultsIn this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average.ConclusionTransient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.

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Human interleukin 2 (IL-2) and human IL-2 mutant proteins, with artificially introduced Nglycosylation or O-glycosylation sites, have been expressed in a lepidopteran cell line (Sf21, Spodopteru frugiperdu) using recombinant baculovirus vectors. Only approximately 25 % of the total recombinant IL-2 protein synthesized by Sf21 cells was secreted into the culture medium. Significant N-terminal truncations were detected in the secreted polypeptides (up to 85% of the molecules). Alanine and proline were absent in the major truncated forms; the first 3-5 amino acids were also absent in a small proportion of the purified proteins.The introduction of potential artificial 0-glycosylation peptide sequences (. . GGKAPTPPPK . .), to the C-terminus or between positions 80 and 81 of the IL-2 polypeptide chain, resulted in the secretion of unglycosylated and 0-glycosylated variant forms. Fast atom bombardment mass spectrometry, compositional analysis and methylation analysis, of the tryptic glycopeptide APTPPPK, revealed the presence of either GalNAc or the disaccharide Gal@ -3)GalNAc as the only carbohydrate constituents attached exclusively to Thr in this peptide, in a specific ratio for each individual IL-2 mutant protein. The Galwl -3)GalNAc protein forms could be partially altered in vitro to mammalian-type glycoforms by porcine liver P-galactoside a-2,3-sialyltransferase in the presence of CMP-N-acetylneuraminic acid.An IL-2 mutant form, with an 11-amino-acid peptide of human interferon$ at position 4, which includes its only N-glycosylation site, had exclusively truncated proximally fucosylated oligomannosidic glycans ; Man,GlcNAc[Fuc(al -6)IGlcNAc or Man2GlcNAc[Fuc(al-6)]GlcNAc structures, in a ratio of 3 : 1, were detected in the secreted proteins. No evidence was obtained for the presence of secreted proteins with complex oligosaccharide chains, irrespective of the cell-culture conditions used or the harvesting time, for infected cells with recombinant baculovirus constructs.In recent years the baculovirus expression system has been increasingly used for the high-level expression of several foreign proteins (see Luckow and Summers, 1988, for a review). In principle, insect cells are capable of most of the posttranslational modifications found in the polypeptides synthesized by mammalian cells (Luckow and Summers, 1988). However, the purified proteins have been thoroughly Abbreviations. AcNPV, Autographu californica nuclear polyhedrosis virus ; FAB, fast atom bombardment; HPAE-PAD, highpH anion-exchange chromatography with pulsed amperometric detection; IFN-P, interferon P; IL-2, interleukin 2; IL-Mu, interleukin variant protein; Sf, Spodopteru frugiperdu cell line; Peptide-N4-(Nacetyl-P-glucosaminy1)asparagine amidase, N-glycosidase F.Enzymes. P-Galactoside a-2,3-sialyltransferase (EC 2.4.99.4); Peptide-N4-(N-acetyl-P-glucosaminyl)asparagine amidase (EC 3.5.1.52).Note. A preliminary account of this work has been presented at the "Workshop on Baculovirus and Recombinant Protein Production Processes", March 29-April 1...