Crystallization and preliminary X-ray crystallographic analysis of a full-length active form of the Cry4Ba toxin fromBacillus thuringiensis

Abstract

To obtain a complete structure of the Bacillus thuringiensis Cry4Ba mosquito-larvicidal protein, a 65 kDa functional form of the Cry4Ba-R203Q mutant toxin was generated for crystallization by eliminating the tryptic cleavage site at Arg203. The 65 kDa trypsin-resistant fragment was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals belonged to the rhombohedral space group R32, with unit-cell parameters a = b = 184.62, c = 187.36 A. Diffraction data were collected to at least 2.07 A resolution using synchrotron radiation and gave a data set with an overall R(merge) of 9.1% and a completeness of 99.9%. Preliminary analysis indicated that the asymmetric unit contained one molecule of the active full-length mutant, with a V(M) coefficient and solvent content of 4.33 A(3) Da(-1) and 71%, respectively.

Potential Prepore Trimer Formation by the Bacillus thuringiensis Mosquito-specific Toxin

“…Toxin Preparations-The 130-kDa Cry4Ba-R203Q mutant protoxin in which one trypsin cleavage site at Arg 203 was removed, therefore producing a 65-kDa active fragment upon trypsin digestion and retaining high Aedes larval toxicity (25), was overexpressed as cytoplasmic inclusions in Escherichia coli JM109 upon isopropyl 1-thio-␤-D-galactopyranoside induction. Toxin activation was done by digesting the protoxin presolubilized in carbonate buffer (50 mM Na 2 CO 3 /NaHCO 3 (pH 9.0)) with trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated, Sigma) as described elsewhere (25).…”

“…Toxin activation was done by digesting the protoxin presolubilized in carbonate buffer (50 mM Na 2 CO 3 /NaHCO 3 (pH 9.0)) with trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated, Sigma) as described elsewhere (25). The 65-kDa activated toxin was purified via FPLC using Superdex 200 or Superose 12 HR10/30 and concentrated to 1-5 mg/ml by ultrafiltration.…”

The C-Terminal Domain of the Bacillus thuringiensis Cry4Ba Mosquito-Specific Toxin Serves as a Potential Membrane Anchor

“…Upon ingestion by susceptible insect larvae, Cry toxins, which are initially produced as protoxin inclusions, are solubilized in the larval midgut lumen (highly alkaline for dipteran and lepidopteran larvae) prior to proteolytic activation by gut proteases to yield active toxins of ~65 kDa [ 1 , 5 ]. The activated Cry toxins display a wedge-shaped architecture with three structurally distinctive domains [ 6 , 7 , 8 , 9 ]: an N-terminal α-helical bundle (DI), a β-sheet prism (DII) and a C-terminal β-sheet sandwich (DIII) (see Figure 1 ). In particular, DI and DII of numerous Cry toxins have been evidently demonstrated to be responsible for membrane-inserted pore formation and target receptor recognition, respectively [ 10 , 11 ].…”

Two specific membrane-bound aminopeptidase N isoforms from Aedes aegypti larvae serve as functional receptors for the Bacillus thuringiensis Cry4Ba toxin implicating counterpart specificity

“…Cry4Ba-R203Q mutant toxin (one tryptic cleavage site was eliminated by substitution at Arg 203 with Gln) which still retains high larvicidal activity [22] was used in this study. E. coli strain JM109 expressing the R203Q mutant was grown in LB medium containing 100 mg/mL ampicillin at 37 C until OD 600 reached 0.3e0.6.…”