A chicken bioreactor for efficient production of functional cytokines.

BACKGROUND: The global market for protein drugs has the highest compound annual growth rate of any pharmaceutical class but their availability, especially outside of the US market, is compromised by the high cost of manufacture and validation compared to traditional chemical drugs. Improvements in transgenic technologies allow valuable proteins to be produced by genetically-modified animals; several therapeutic proteins from such animal bioreactors are already on the market after successful clinical trials and regulatory approval. Chickens have lagged behind mammals in bioreactor development, despite a number of potential advantages, due to the historic difficulty in producing transgenic birds, but the production of therapeutic proteins in egg white of transgenic chickens would substantially lower costs across the entire production cycle compared to traditional cell culture-based production systems. This could lead to more affordable treatments and wider markets, including in developing countries and for animal health applications. RESULTS: Here we report the efficient generation of new transgenic chicken lines to optimize protein production in eggs. As proof-of-concept, we describe the expression, purification and functional characterization of three pharmaceutical proteins, the human cytokine interferon α2a and two species-specific Fc fusions of the cytokine CSF1. CONCLUSION: Our work optimizes and validates a transgenic chicken system for the cost-effective production of pure, high quality, biologically active protein for therapeutics and other applications.

The intracellular interactions of the L1 family of cell adhesion molecules.

The L1 family of CAMs (cell adhesion molecules) has long aroused the interest of researchers, but primarily the extracellular interactions of these proteins have been elucidated. More recently, attention has turned to the intracellular signalling potentiated by transmembrane proteins and the cytoplasmic proteins with which they can interact. The present review brings up to date the current body of published knowledge for the intracellular interactions of L1-CAM family proteins and the potential importance of these interactions for the mechanisms of L1-CAM action.

A functional FERM domain binding motif in neurofascin.

The L1 family of transmembrane cell adhesion receptors are involved in the development of the nervous system and consist of L1, neuron-glial-related cell adhesion molecule and neurofascin. All three receptors have a short cytoplasmic tail which is known to bind to the cytoskeletal associated protein ankyrin. Ezrin is a cytoplasmic binding protein known to link plasma membrane proteins to the cytoskeleton and has been shown to be a binding partner for L1. Here we show that neurofascin can also interact directly with ezrin. However, the mechanism of interaction of L1 and neurofascin with ezrin is by different mechanisms. We also show that the neurofascin isoform, Nfasc155, co-localizes with ezrin in transfected HEK293 cells but also in interdigitating Schwann cells at the node of Ranvier.

A novel 4.1 ezrin radixin moesin (FERM)-containing protein, 'Willin'.

The 4.1 superfamily of proteins contain a 4.1 Ezrin Radixin Moesin (FERM) domain and are described as linking the cytoskeleton with the plasma membrane. Here, we describe a new FERM domain-containing protein called Willin. Willin has a recognizable FERM domain within its N-terminus and is capable of binding phospholipids. Its intra-cellular distribution can be cytoplasmic or at the plasma membrane where it can co-localize with actin. However, the plasma membrane location of Willin is not influenced by cytochalasin D induced actin disruption but it is induced by the addition of epidermal growth factor.

Metal binding Asp-120 in metallo-beta-lactamase L1 from Stenotrophomonas maltophilia plays a crucial role in catalysis.

Metallo-beta-lactamase L1 from Stenotrophomonas maltophilia is a dinuclear Zn(II) enzyme that contains a metal-binding aspartic acid in a position to potentially play an important role in catalysis. The presence of this metal-binding aspartic acid appears to be common to most dinuclear, metal-containing, hydrolytic enzymes; particularly those with a beta-lactamase fold. In an effort to probe the catalytic and metal-binding role of Asp-120 in L1, three site-directed mutants (D120C, D120N, and D120S) were prepared and characterized using metal analyses, circular dichroism spectroscopy, and presteady-state and steady-state kinetics. The D120C, D120N, and D120S mutants were shown to bind 1.6 +/- 0.2, 1.8 +/- 0.2, and 1.1 +/- 0.2 mol of Zn(II) per monomer, respectively. The mutants exhibited 10- to 1000-fold drops in kcat values as compared with wild-type L1, and a general trend of activity, wild-type > D120N > D120C and D120S, was observed for all substrates tested. Solvent isotope and pH dependence studies indicate one or more protons in flight, with pKa values outside the range of pH 5-10 (except D120N), during a rate-limiting step for all the enzymes. These data demonstrate that Asp-120 is crucial for L1 to bind its full complement of Zn(II) and subsequently for proper substrate binding to the enzyme. This work also confirms that Asp-120 plays a significant role in catalysis, presumably via hydrogen bonding with water, assisting in formation of the bridging hydroxide/water, and a rate-limiting proton transfer in the hydrolysis reaction.