Expression And Identification Of Huwentoxin-ⅩⅧal In Prokaryotic System

Ornithoctonus huwena, a rare poisonous spider in our country, narrowly distributed in virgin forests of China’s GuangXi province, Yunnan province and some south-east Asian countries such as Vietnam. Its venom is a complex mixture composed of many biological active ingredients; 90 proteins and 47 peptides have been identified from crude venom with the method of proteomics and peptidomics.But most of them belong to the high abundant components. Through proteomics and peptidomics combined with the transcriptomic methods, our previous study grouped the ingredients except for HWTX-Ⅺ and the HWTX-ⅩⅢ, into eight superfamilies: HWTX-Ⅰ, HWTX-Ⅱ, HWTX-Ⅹ, HWTX-ⅩⅣ, HWTX-ⅩⅤ, HWTX-ⅩⅥ, HWTX-ⅩⅦ and HWTX-ⅩⅧ. Most peptides contain abundant cysteine residues, hence their name cystein knot toxin (CKT).HWTX-ⅩⅧa1, a member of HWTX-ⅩⅧ super family, is made up of 64 amino acid residues, containing four pairs of disulfide bond, with pI of 7.72/Mv, and average molecular weight of 6433.29 Da. It is very low abundance in crude venom. The natural peptide was identified in crude venom proteomics and transcriptomics individually. However, due to its low abundance, it is difficult to get enough natural peptide for functional study.Furthermore, the chemical synthesis is a challenge for this peptide with 64 aa. Additionally, the previous work of this laboratory showed that the yeast expression system is also not working for this peptide. Finally, we turned into E. coli prokaryotic expression system.In this study, plasmid pET-32 a (+) and E. coli BL21-RIPL strains have been used. HWTX-VIIIal gene were cloned into pET-32 a (+) plasmid, then transformed into the E. coli BL21-RIPL competence cell. When IPTG concentration is between 0.6 to 0.8 mM, the quantity of fusion reached the highest level. Recombinant HWTX-VIIIal peptide containing Trx-and 6 x His-tag were affinity purified with Ni-NTA beads.For the removal of tag, two strategies have been designed:1. A Factor Xa protease cleavage site (Ile-Glu-Gly-Arg↓)was introduced between the tag and the target gene. (1) The cleavage site is directly introduced without hinged linker; (2) An additional hinge fragment (Gly-Thr-Gly-Gly-Gly-Ser-Gly) was inserted before the Factor Xa protease recognition site. As the result, for recombinant peptide without hinge, the solubility of peptide is low. It was easy to form aggregate and digestion efficiency is also not good. In contrast, the introducing of a flexible short peptide sequence facilitated the cleavage.2. A formic acid cutting site (Asp↓Pro) was introduced between the label protein and the target protein site. The cost of formic acid is low, but will introduce additional Pro in the N-terminal of target peptide. Our experimental results showed that the pattern of digestion products is complex after formic acid treatment. It may bring trouble to further purification.According to above data, we adopted the scheme 1(2) in the further study. The separation pattern of recombinant peptide after cleavage on 16% Tricine-SDS gel clearly showed the bands of recombination polypeptides, released tag peptides, tag-free target.In order to effectively remove the recombination polypeptides and the released tag peptides(both containing 6×His tag),we have been used Ni-NTA agarose for adsorption. However, the adsorption is not complete and also specific. Then, the ion exchange chromatography was used instead. The electrophoresis map showed that there were peptide bands with molecular weight between 10 and 3.5 KDa(which containing our target peptides) However, these low molecular weight components were only indicated in the flow-through of SP strong cation exchange column(20 mM phosphate buffer, pH 6.5). Further MALDI-TOF mass-spectrometry analysis uncovered a peak with MW 6433.1 Da, which is consistent to the theoritical value of HWTX-Ⅷa. The slight difference on molecular weight may indicate the difference between unfolding peptide and natural peptides.Since most of enzyme cutting product with molecular weight more than 10 KDa could be removed by SP sepharose, this step were used for crude separation.However, in the SP-sepharose flow-through, a high peak with MW 5007 Da also showed in the MS analysis. It prompts us to further purify it through more precise way.Reverse phase high pressure liquid chromatography were tried for further purification. However, there is no any elution peak on C18 and C4 RP-HPLC column even at 100% ACN. It showed the difference between the HWTX-Ⅷal in unfolding state and natural state.Our work is the necessary step for the re-naturation and finally functional study of HWTX-XVIIIal.It also provided a approach to for expression of larger toxic peptide.