Metallo--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 -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 k cat 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 pK a 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.
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.
BackgroundThe 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.ResultsHere 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.ConclusionOur 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.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0495-1) contains supplementary material, which is available to authorized users.
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