Expression and refolding of bioactive α-bungarotoxin V31 in E. coli.

In order to obtain bioactive α-bungarotoxin (αBtx) using recombinant protein technique, a codon-optimized synthetic gene was expressed in fusion with the N-terminal 10-His-tag and C-terminal Strep-tag in Escherichia coli. Further optimization through site-directed mutagenesis enabled moderate expression of the protein without the N-terminal His-tag or the C-terminal Strep-tag. Two such recombinant αBtx (rαBtx) were obtained, both with an additional methionine and a glycine at the N-terminal and one with (G4S1)2-Strep-tag at the C-terminal. The rαBtx proteins were refolded using a novel protocol, which efficiently produced final products with activity similar to its natural counterpart. The protocol could easily be scale up, which produced 0.3-1mg of pure and highly active rαBtx per liter of E. coli culture.

[Cloning of alpha-bungarotoxin gene and its prokaryotic expression as a non-fusion protein].

On the basis of the reported amino acid sequence of alpha-bungarotoxin (alpha-BGT), DNA sequence of alpha-BGT was deduced and fourteen partially complementary oligonucleotides were designed and synthesized. A plasmid carrying the coding region of alpha-BGT was obtained by primer extension, PCR and ligation with pMD-18-T. The target fragment was digested with Xba I and EcoR I, recovered and ligated with pET28a(+). The resultant expression vector was transformed into BL21 (DE3), BL21 (DE3) Codon plus, and BL21 (DE3) plysS, respectively. Recombinant alpha-BGT was expressed in BL21 (DE3) and was analyzed by 15% Tris/tricine SDS-PAGE. The result showed that the recombinant protein, mostly found in inclusion bodies, accounted for 11.98% of the total bacterial lysate. The expression capacity could be increased to 16.28% by optimizing expression conditions. Western blotting results showed that the expressed protein had similar immunogenicity with the natural alpha-BGT protein purified from the venom of Krait Bungarus spp. In vivo toxicity assay of purified and renatured proteins in mice showed that LD50 was about 1.28 microg/g.

Cloning and functional expression of B chains of beta-bungarotoxins from Bungarus multicinctus (Taiwan banded krait).

The cDNA species encoding the B chains (B1 and B2) of beta-bungarotoxins (beta-Bgt) were constructed from the cellular RNA isolated from the venom glands of Bungarus multicinctus (Taiwan banded krait). The deduced amino acid sequences of the B chains were different from those determined previously by a protein sequencing technique. One additional Arg residue is inserted between Val-19 and Arg-20 of the B1 chain. Similarly the insertion of one additional Val residue between Val-19 and Arg-20 of the B2 chain is noted. Thus the B chains should comprise 61 amino acid residues. Moreover, the residues at positions 44-46 are Gly-Asn-His, in contrast with a previous result showing the sequence His-Gly-Asn. Instead of Asp, the residues at positions 41 and 43 are Asn. The B chain was subcloned into the expression vector pET-32a(+) and transformed into Escherichia coli strain BL21(DE3). The recombinant B chain was expressed as a fusion protein and purified on a His-Bind resin column. The yield of affinity-purified fusion protein was increased markedly by replacing Cys-55 of the B chain with Ser. However, the isolated B(C55S) chain became insoluble in aqueous solution after removal of the fused protein from the affinity-purified product, suggesting that protein-protein interactions might be crucial for stabilizing the structure of the B chain. The B(C55S) chain fusion protein showed activity in blocking the voltage-dependent K+ channel, but did not inhibit the binding of beta-Bgt to synaptosomal membranes. These results, together with the finding that modification of His-48 of the A chain of beta-Bgt caused a marked decrease in the ability to bind toxin to its acceptor proteins, suggest that the B chain is involved in the K+ channel blocking action observed with beta-Bgt, and that the binding of beta-Bgt to neuronal receptors is not heavily dependent on the B chain.

cDNA sequence analysis and expression of kappa-bungarotoxin from Taiwan banded krait.

The cDNAs encoding kappa-bungarotoxin was constructed from the cellular RNA isolated from the venom glands of Bungarus multicinctus by reverse transcription-polymerase chain reaction. A high degree of nucleotide sequence homology was observed between kappa-bungarotoxin and other kappa-neurotoxins. The kappa-bungarotoxin was subcloned into the expression vector pET32a(+) and transformed into BL21(DE3) E. coli strain. The recombinant toxin was expressed as a fusion protein. Recombinant kappa-bungarotoxin was separated from the fused protein by cleavage with CNBr and purified by reversed phase high performance liquid chromatography. In addition to kappa-bungarotoxin, the cDNA fragment encoding kappa3-bungarotoxin was also found in the cDNA mixtures prepared from the cellular RNA of the venom glands of the same snake. This result suggests that the venom glands of Taiwanese B. multicinctus should secrete at least two kinds of kappa-neurotoxins.

Facile production of native-like kappa-bungarotoxin in yeast: an enhanced system for the production of a neuronal nicotinic acetylcholine receptor probe.

Research on the mammalian central nervous system had been hindered by the limited number and meager supply of naturally occurring toxins that can be used as pharmacological reagents. The kappa-neurotoxins in particular are not found abundantly in nature and are difficult to obtain and isolate in quantities sufficient for research purposes. Here we report the expression and isolation of relatively large quantities of the kappa-neurotoxin, kappa-bungarotoxin, in an active form using a yeast, Pichia pastoris, expression system. The resultant product of the expression system has a short amino-terminal amino acid extension relative to venom-derived kappa-bungarotoxin, but is equivalent to the native toxin in physical and biological properties, as judged by the CD spectra, the ability to form dimers in solution, and the activity on chick ciliary ganglia. The yeast system produces approximately 0.2 mg from a 2 liter culture and the purification takes approximately 2 days. In contrast, E. coli, the only other available expression system for this toxin, produces one-fifth to one-half as much active material from a 5 liter high-density fermentation and the resulting protein takes over a week to purify. No high mol. wt disulfide-bonded aggregates were found in the yeast expression system product, indicating that the product is that of a biologically assisted folding process. This has significant implications not only for the efficient production of native toxin but also for the production of mutant proteins to study the structure-function relationship in these proteins.

Functional expression and site-directed mutagenesis of a synthetic gene for alpha-bungarotoxin.

In order to explore the structure-function relationships of the curare mimetic alpha-neurotoxins we have constructed and cloned a synthetic gene for Bungarus multicinctus alpha-bungarotoxin which is expressed in Escherichia coli. The recombinant alpha-bungarotoxin is expressed as a fusion protein with alpha-bungarotoxin linked to the COOH-terminal end of the T7 Gene 9-encoded coat protein. After treatment of the fusion protein with Factor Xa protease, a recombinant alpha-bungarotoxin is released that co-migrates with authentic alpha-bungarotoxin upon reverse-phase high performance liquid chromatography and ion-exchange chromatography. Final yields of active recombinant alpha-bungarotoxin were about 0.4 mg/liter of starting bacterial culture. The recombinant alpha-bungarotoxin contains 10 additional residues linked to the NH2-terminal Ile of the alpha-bungarotoxin sequence due apparently to the inaccessibility of the engineered cleavage site to Factor Xa. Nevertheless, the recombinant alpha-bungarotoxin is capable of binding to the nicotinic acetylcholine receptor with an apparent affinity that is only decreased approximately 1.7-fold from that of authentic alpha-bungarotoxin. Alanine substitution of a residue, Asp30, highly conserved among alpha-neurotoxins and previously suggested to play a key role in receptor recognition, resulted in a recombinant alpha-bungarotoxin whose receptor binding activity is indistinguishable from authentic alpha-bungarotoxin.

Synthesis and expression in Escherichia coli of a gene for kappa-bungarotoxin.

A gene which codes for the 66-residue polypeptide of kappa-bungarotoxin has been chemically synthesized by linking together 3 synthetic double-stranded oligonucleotides in a bacterial plasmid. The synthesis incorporated six unique silent restriction sites spaced throughout the gene for use in cassette mutagenesis. Direct expression of the kappa-bungarotoxin polypeptide by itself in Escherichia coli failed to result in a stable product. The toxin polypeptide was stabilized and expressed in E. coli as part of a fusion protein with rat intestinal fatty acid binding protein under control of the nalidixic acid inducible recA promoter. Two fusion protein constructs were prepared that differed only in the cleavage site between the fatty acid binding protein and the toxin polypeptide. One contained a factor Xa cleavage site, and the other, since the toxin itself is devoid of methionine, contained a methionyl residue that served as a cyanogen bromide cleavage site. The fusion proteins were isolated by ion-exchange chromatography and reverse-phase HPLC. The construct containing the factor Xa cleavage site could not be cleaved under nondenaturing conditions. On the other hand, kappa-bungarotoxin was efficiently cleaved from the methionyl fusion protein with CNBr. The toxin polypeptide was isolated by reverse-phase HPLC and ion-exchange chromatography and produced a complete and specific blockade of neuronal nicotinic acetylcholine receptors in chick ciliary ganglia which was indistinguishable from that produced by a comparable amount of venom-purified kappa-bungarotoxin.