How Dextran Sulfate Affects C1-inhibitor Activity: A Model for Polysaccharide Potentiation.

C1-inhibitor is a key inhibitor of the complement and contact activation systems, and mutations in the protein can cause hereditary angioedema. Through an unknown mechanism, polysaccharides can increase C1-inhibitor activity against some of its target proteases. Here we present the crystal structures of the serine protease inhibitor (serpin) domain of active C1-inhibitor by itself and in complex with dextran sulfate.

Recombinant human fibrinogen that produces thick fibrin fibers with increased wound adhesion and clot density.

Human fibrinogen is a biomaterial used in surgical tissue sealants, scaffolding for tissue engineering, and wound healing. Here we report on the post-translational structure and functionality of recombinant human FI (rFI) made at commodity levels in the milk of transgenic dairy cows. Relative to plasma-derived fibrinogen (pdFI), rFI predominantly contained a simplified, neutral carbohydrate structure and >4-fold higher levels of the γ'-chain transcriptional variant that has been reported to bind thrombin and Factor XIII.

Characterization of recombinant human C1 inhibitor secreted in milk of transgenic rabbits.

C1 inhibitor (C1INH) is a single-chain glycoprotein that inhibits activation of the contact system of coagulation and the complement system. C1INH isolated from human blood plasma (pd-hC1INH) is used for the management of hereditary angioedema (HAE), a disease caused by heterozygous deficiency of C1INH, and is a promise for treatment of ischemia-reperfusion injuries like acute myocardial or cerebral infarction. To obtain large quantities of C1INH, recombinant human C1INH (rhC1INH) was expressed in the milk of transgenic rabbits (12 g/l) harboring genomic human C1INH sequences fused to 5' bovine αS(1) casein promoter sequences.

The protein structure of recombinant human lactoferrin produced in the milk of transgenic cows closely matches the structure of human milk-derived lactoferrin.

Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the host defence against infection and excessive inflammation. As the availability of (human milk-derived) natural hLF is limited, alternative means of production of this biopharmaceutical are extensively researched. Here we report the crystal structure of recombinant hLF (rhLF) expressed in the milk of transgenic cows at a resolution of 2.

The role of N-linked glycosylation in the protection of human and bovine lactoferrin against tryptic proteolysis.

Lactoferrin (LF) is an iron-binding glycoprotein of the innate host defence system. To elucidate the role of N-linked glycosylation in protection of LF against proteolysis, we compared the tryptic susceptibility of human LF (hLF) variants from human milk, expressed in human 293(S) cells or in the milk of transgenic mice and cows. The analysis revealed that recombinant hLF (rhLF) with mutations Ile130-->Thr and Gly404-->Cys was about twofold more susceptible than glycosylated and unglycosylated variants with the naturally occurring Ile130 and Gly404.

Analytical cation-exchange chromatography to assess the identity, purity, and N-terminal integrity of human lactoferrin.

Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the innate host defense. The positively charged N-terminal domain of hLF mediates several of its activities by interacting with ligands such as bacterial lipopolysaccharide (LPS), specific receptors, and other proteins. This cationic domain is highly susceptible to limited proteolysis, which impacts on the affinity of hLF for the ligand.

Characterization of monoclonal antibodies against human lactoferrin.

The iron-binding glycoprotein human lactoferrin (hLF) is involved in the host defense against infection and is a modulator of inflammatory reactions. We generated monoclonal antibodies (mAbs) to hLF as tools to assist both structure-function studies and the development of recombinant human lactoferrin for applications in human health care. Binding experiments with ten distinct anti-hLF mAbs to tryptic and recombinant hLF fragments in ELISA and/or on immunoblots revealed that five mAbs bound to conformational epitopes residing in the N-lobe (residues 1 to 334), whereas the other five bound to C-lobe conformational epitopes (residues 335 to 692).