| A distinct feature of the toxin distribution from the black widow spider(L.tredecimguttatus)is their exceptional existence outside the venomous glands such as in its eggs.It is commonly considered that the significance of egg toxins lies in protecting the eggs from some greedy animals and pathogenic microorganisms,thereby improving the survival probability and evolutionary advantage of the spider species.A series of researches including proteomic analyses revealed the strong discrepancy between the molecular bases for toxicities of the eggs and venomous glands,suggesting great potential in the discovery of novel toxins and other bioactive components from the eggs for the development of tool agents and clinical drugs.At present,the mostly used strategy for obtaining desired toxins and other bioactive molecules is direct extraction from natural materials followed by activity screening,which,however,often has limitations of high cost,low yield,and long period,etc.,particularly when the natural material is rare,the material composition is complex,and the desired component is low-abundant.Heterologous expression based on the molecular biological strategy provides a natural extraction-independent efficient alternative to the screening of proteinaceous bioactive components,provided that their full-length encoding gene is known.Owing to the rapid development of the next-generation RNA sequencing technique,it is possible for the transcriptome to be sequenced fast and at low-cost,which provides much information on the full-length gene sequences encoding for the proteins and peptides,including those expressed in such a low abundance that it is impossible for them to be directly purified from the natural materials,which provides the convenience for the natural extraction-independent gene cloning,expression and activity screening of proteins and peptides.In our previous work,we had sequenced the transcriptome of black widow spider eggs and obtained 14,185 unigenes,of which 280 unigenes were demonstrated to encode for the proteins homogenous to known toxins.In the present study,we selected a unigene from the transcriptome that encodes for a peptide with ICK(inhibitor cystine knot)motif,a common feature of many peptide neurotoxins,to clone and heterologously express in order to prepare the toxin without direct extraction from the natural material.We first extracted the total RNA from eggs of the spider,and then,a cDNA library was constructed by reverse transcription of the total RNA.The DNA sequence of interest was amplified through 3' RACE combined with nested PCR.The PCR product was ligated into pMD19-T vector and transformed into E.coli DH5a.After that we amplified the whole CDS region of the sequence from the recombinant T-vector using a pair of expression primers with special modifications and cloned the products into pET32a.Protein expression was successfully induced by 0.5 mM IPTG at 27 oC.Taking advantages of the Ni-NTA agarose,the fusion protein was affinity purified to electrophoretic purity very smoothly.To obtain the pure peptide of interest,the fusion protein was digested with recombinant bovine enterokinase,followed by RP-HPLC.It was shown by ESI(electrospray ionization)mass spectrometry that the monoisotopic mass of the peptide was 6195.61 Da,while the theoretical monoisotopic mass of the reduced peptide was 6205.67 Da,suggesting that the ten cysteine residues in the recombinant peptide formed five disulfide bonds.Patch clamp experiments indicated that the peptide at a concentration of 2 ?mol/L could inhibit about 37%of the Nav channel currents on ND 7/23 cell,and,however,no effects on Nav channel currents on the DUM neuron of Periplaneta americana were observed even when the neuron was treated with 10 ?mol/L of the peptide.So the peptide was demonstrated to be a toxin selectively interacting with the Nav channels on mammalian neurons.Based on the order in which the peptide was found and characterized in the black widow spider eggs,this peptide was named Latroeggtoxin-VI.This work not only laid a foundation for further researches on the structure-function and potential application of the peptide toxin,but also provided a reference for screening of proteinaceous bioactive components using a natural extraction-independent strategy. |
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