Ribonuclease H - selenomethionyl protein phased at 2 A resolution by MAD

The multiple isomorphous replacement method is limited when protein does not bind heavy atoms or when binding induces nonisomorphism between native and derivatized crystals. An alternative method for heavy-atom derivatisation is substitution of methionine by selenomethionine. It offers a general method for introduction of anomalous scatters into cloned proteins. In 1957 Cohen and Cowie discovered that an Escherichia coli strain, which is auxotrophic for methionine, grow in a medium containing selenomethionine [22]. W. Hendrickson and co-workers, thirty years later, show that selenium is a useful anomalous scatterer [23]. They solved the structure of selenobiotyl streptavidin [24] and of selenomethionyl proteins [25], using multiwavelength anomalous dispersion (MAD). Preparation of selenomethionine-containing protein is simply and relatvely easy to perform.

Transformation

The cells producing the selenomethionine-labelled proteins should be auxotrophic for methionine and will be grown in selenomethioninenmedium. The auxotrophic met-cells in most cases can be transformed with the plasmid producing the cloned protein. Esherichia coli strains differ in their tolerance to selenomethionine. The DL41 strain, constructed by LeMaster [26] is widely used as a general host for plasmid transformation. Transformation is carried out by standard procedures [27]

Transduction

Transduction is usually used when a particular Escherichia coli strain need to be preserved. It is easy to introduce a met- mutation by transduction and inactivate a homologous gene on the bacterial chromosome. A new characteristic, such as methionine auxotrophy (met-), is incorporated into the strain genome via bacteriophage (e.g. P1vir). The recipient strain becomes auxotrophic for methionine. Optimally, the inactivated met allele and recipient strain will have two types of antibiotics resistance. The transduced cells can be selected on a medium containing both antibiotics (one for the expression plasmid and other for met auxotrophy). Tranduction is described in detail in Protocol 1 (from reference [28])

Usually, bacterial cells grow more slowly in selenomethionine medium. The stationary phase is reached at a lower final cell density. Cells grown in selenomethionine tend to stay in stationary phase. and need a small amount of rich medium to get out of stationary phase. A small amout (2-5%v/v) of LB medium in starter culture (or 5 ml culture in rich medium - LB to inoculate starter culture) will provide sufficient methionine to revive the cells from stationary phase. The starter culture (about 100 ml) will be used to inoculate a 10-20 liter fermenter. The final dilution of the initial methionine should be as high as possible, since during fermentation methionine is incorporated in preference to selenomethionine. This amount of rich medium (LB) will be a compromise between a better growth rate and complete methionine substitution. For each particular strain the amount of rich medium in the starter culture and selenomethionine concentration in the fermentation medium should be determined. An example of medium and procedure that could be used for auxotrophic cell growth is described in Protocol 2 (from reference [28])

Inhibition of the methionine biosynthesis pathway is a recently developed technique. It does not require auxotrophic met- strains and is based on the blocking of methionine biosynthesis, by inhibiting aspartokinases in the presence of high concentration of isoleucine, lysine and threonine. The nonauxotrophic strain is growing in medium containing a high concentration of aminoacids known to inhibit methionine biosynthesis and without methionine (replaced by abundance of selenomethionine). This method does not require a new expression vector and is potentialy applicable to any prokaryotic strain. There are some applications of this procedure: UDP-N-acetylenopyruvylglucosamine reductase [29], FKBP-12 [30], 9kDa protein of the signal recognition particle [31]. Example of the methionine pathway inhibition procedure is shown in Protocol 3 (adapted from reference [28]).

Animal cells are naturally auxotrophic for methionine. Therefore eukaryotic cells growing in selenomethionine medium show very good incorporation of the substitute amino acids. The selenomethionyl human chorionic gonadotropin (hCG) was produced using two systems:

• baculovirus system [32] with 92 % substitution rate (measured by amino acid analysis);
• chinese hamster ovary [33]. with about 84%incorporation of selenomethionine (measured as above).

Selenomethionyl protein is much more sensitive to oxidation than natural protein. If selenium atoms are on the surface of the protein molecule they can alter protein solubility and hydrophobicity. Usually it is more hydrophobic and less soluble. These properties require some modifications to the normal purification. To avoid oxidation of selenomethionine all buffers should be degassed. Buffers should include a reducing reagent such as dithiothreitol (DTT) and a chelator such as Ethylene Diamine Tetraacetic Acid (EDTA) to remove traces of metals that could catalyse oxidation.