Preparing SeMet Derivatives

From Maciej Kozak:

Introduction

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.

Expression in Prokaryotes

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])

Cell growth

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])

Methionine Pathway inhibition

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]).

Expression in Eukaryotes

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).

Purification of Selenomethionyl Proteins

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.

From Expressions Systems:

Protocol for Selenomethionine Incorporation: This protocol assumes standard cell culture operations which are: suspension cells, grown either in shake flasks, spinner vessels or Wave units, utilizing a “complete medium” recommended for standard BEVS. Cells are assumed to be high viability, ( > 98% ) demonstrating robust growth rates and little to no cell clumps.

Preparation of methionine-free medium cell stock. Transfer standard suspension cell stock, ( i.e., Sf9, Sf21, or Tni cells ) in mid-log growth, from standard cell culture medium to methionine-free medium. Use 15 mls of culture medium, cells should be at a density of 2-3 x 106 per ml, to inoculate 30 mls of methionine-free medium for a final 45 ml of culture. Grow cells exclusively in methionine-free medium for 2 passages, allowing cells to grow to a maximum of 4 x 106 per ml, 2 x 106 if Sf21 cells are used.

Selenomethionine Incorporation. Split culture back to 7 x 105 in methionine-free media; wait 24 hours and infect with standard MOI. Add DL-selenomethionine at 100mgs per liter, at various time intervals, beginning no earlier than 8 hours after time of infection, then at sequential 24 hour intervals after infection up to harvest time and compare to when first additions were made at 24, 48, 72 hour intervals after infection Harvest at day 3 or day 4 or at 50% viability.

From Robert Frank Standaert's (Cornell University) Thesis:

Consultation of a biochemistry textbook indicated that threonine, isoleucine, and lysine inhibit methionine biosynthesis in E. coli by inhibiting aspartokinases, and that phenylalanine and leucine are synergistic with lysine. Incorporation of this information into a strategy for SeMet labeling was simple. E. coli XA90 were grown in 1 L of M9 medium to mid-log phase (A600=0.6), and 60 mg of SeMet (sigma) along with 100 mg each of threonine, lysine hydrochloride, and phenylalanine and 50 mg each of leucine, isoleucine, and valine were added as solids to the growing culture. After another 15 min, IPTG was added to 1 mM, and the procedure was completed as usual.Purification was performed essentially as for native protein, except that DTT at a concentration of 5-10 mM was maintained throughout on the expectation that SeMet would be sensitive to oxidation. The yield of protein was 10 mg, about what would be obtained from a normal prep.