1. Cloning and functional expression of a synthetic gene encoding huwentoxin-I, a neurotoxin from the Chinese bird spider (Selenocosmia huwena)
M Li, L Y Li, X Wu, S P Liang Toxicon. 2000 Feb;38(2):153-62. doi: 10.1016/s0041-0101(99)00123-3.
Cloning and functional expression of a synthetic gene encoding huwentoxin-I, a neurotoxin from the Chinese bird spider Selenocosmia huwena. A gene encoding huwentoxin-I, a peptide neurotoxin consisted of 33 amino acid residues from the venom of the Chinese bird spider Selenocosmia huwena, was designed, synthesized and expressed in Escherichia coli as a hybrid protein fused with glutathione S-transferase at the N-terminal. The fusion protein was purified by GSH-Sepharose 4B affinity column chromatography and cleaved by thrombin to release the toxin peptide. The amino acid sequence of the recombinant toxin was consistent with the designed one by sequence determination and MALDI-TOF mass analysis, suggesting that the recombinant huwentoxin-I produced the same expression product as the native one. After reduction and renaturation, the biological activity of the recombinant toxin was identical with that of the native huwentoxin-I by electrophysiological method.
2. Generally applicable, convenient solid-phase synthesis and receptor affinities of octreotide analogs
W B Edwards, C G Fields, C J Anderson, T S Pajeau, M J Welch, G B Fields J Med Chem. 1994 Oct 28;37(22):3749-57. doi: 10.1021/jm00048a011.
Octreotide, an analogue of the hormone somatostatin, has applications as a therapeutic and imaging agent for somatostatin-positive tumors. We have developed a generally applicable, convenient stepwise solid-phase synthetic protocol for octreotide (D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-threoninol). [Cys(Acm)2,D-Trp(Boc)4,Lys(Boc)5,Thr(tBu)6,Cys(Acm)7, des(threoninol)]-octreotide was assembled by Fmoc solid-phase synthesis and the intramolecular disulfide bond formed by treatment of the resin-bound peptide with thallium trifluoroacetate [Tl(Tfa)3]. Side-chain protection of Trp by the Boc group was found to preserve Trp integrity during Tl(Tfa)3 treatment. The protected peptide was cleaved from the resin by aminolysis with threoninol and purified by semipreparative RP-HPLC. Isolated [D-Trp(Boc)4,Lys(Boc)5,Thr(tBu)6]octreotide had the correct molecular mass ([M+H]+ = 1275 Da) and sequence and was obtained in 14% yield at > 98% purity. [D-Trp(Boc)4,Lys(Boc)5,Thr(tBu)6]octreotide was utilized for the solution-phase synthesis of CPTA-D-Phe1-octreotide, where CPTA is 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)methyl]benzoic acid. Cyclic dianhydride of diethylenetriaminepentaacetic acid (DTPA) was coupled to a portion of the protected peptide-resin following disulfide bond formation. The DTPA-conjugated, side-chain-protected peptide was cleaved from the resin by aminolysis with threoninol, side-chain deprotected with trifluoroacetic acid, and purified by semipreparative RP-HPLC. The isolated DTPA-D-Phe1-octreotide had the correct molecular mass ([M+H]+ = 1395 Da) and was obtained in 5% yield at > 90% purity. The efficiency of aminolysis was partially dependent upon the linkage between 4-(hydroxymethyl)phenoxy (HMP) handles and the resin and/or resin particle size. The somatostatin receptor binding affinities of synthetic DTPA-D-Phe1-octreotide and CPTA-D-Phe1-octreotide to AtT-20 mouse pituitary carcinoma cell membranes were examined by labeling with 111In and 64Cu, respectively, and performing Scatchard analyses. The dissociation constant (Kd) for our synthetic [111In]DTPA-D-Phe1-octreotide was 4.31 nM, which is comparable to a Kd = 5.57 nM obtained with commercially available DTPA-D-Phe1-octreotide. The Kd for [64Cu]CPTA-D-Phe1-octreotide was 78.5 pM. On the basis of the criteria of molecular mass, RP-HPLC elution time, sequence analysis, and somatostatin receptor binding affinity, our synthetic octreotide is identical to commercially available octreotide. The aminolysis protocol used here has distinct advantages over either reductive cleavage or preformed linker methods described previously for the preparation of octreotide.
3. The investigation of Fmoc-cysteine derivatives in solid phase peptide synthesis
S N McCurdy Pept Res. 1989 Jan-Feb;2(1):147-52.
Fmoc-Cys(t-Bu)-OH, Fmoc-Cys(Acm)-OH, and Fmoc-Cys(Trt)-OH exhibit excellent synthesis characteristics when used in Fmoc solid phase peptide synthesis on the Applied Biosystems Model 431A peptide synthesizer. The actual 5% scavenger mixture will vary according to the particular amino acid residues present. As was previously mentioned, an anisole/ethanedithiol/ethylmethylsulfide mixture (3:1:1) works well as a general scavenger solution for TFA cleavage of Fmoc synthesized peptide resins. It also may be possible to use lower acid (TFA) concentrations. The syntheses and workups of the peptide Somatostatin utilizing these derivatives demonstrate the ease of using these cysteine derivatives with the Fmoc chemistry approach. The use of either the t-Bu or the Acm moiety produces a peptide containing protected thiol groups after cleavage with 95% TFA. The Fmoc-Cys(Trt)-OH derivative is efficiently deprotected using 95% TFA. This investigation should provide further insight into synthesis options and cleavage protocols when working with cysteine-containing peptides.
