Omega-conotoxin MⅦA Expressed In Pichia Pastoris Has Strong Bioactivity And Its Polyclonal Antibody Preparation

Cone snails (genus Conus) have more than 500 species. Conotoxins (CTXs), are some bioactive polypeptides, which extract from cone snails. According to a conservative estimate, there are more than 50,000 different conotoxins. So far, only small part of conotoxins has been characterized. They are termed as α-, ω-, μ-, σ-types by their different action on ion channels. ω-conotoxins are blockers of voltage-dependent calcium channels, they have a high analgesic effect and have been widely used in neuroscience research. Among the family of co-conotoxins, ω-CTX MⅦA is a higher polar peptide containing six cysteine residues linked by three disulfide bridges and six basic amino acid residues.Three rigid intramolecular disulfide bridges between the conserved cysteine residues (Cl and C16, C8 and C20, and C15 and C25) allow the peptide to fold into a compact globular conformation containing four different loops. Several lines of evidence indicate that the backbone is crucial for its biological function. ω-CTX MⅦA can selectively and specificly inhibit N-type voltage-sensitive calcium channels (VSCCs). Several studies have demonstrated that ω-CTX MⅦA has effects of antinociception and neuroprotection in animal models of global, focal cerebral ischemia and become a commonly used tool for the identification of native N-type calcium channels. The chemical synthetic version of ω-CTX MⅦA (Ziconotide) has been approved for management of severe pains and ischemia by FDA recently.Currently ω-CTX MⅦA used on clinical trials has been obtained by chemicalsynthesis which yield is usually very low and expensive. Encouraged by the double effects of antinociception and neuroprotection, we were interesting in producing co-CTX MVIIA by gene engineering. In this article, we construced the recombinant plasmid pPIC9-CTX and expressed the peptide co-CTX MVIIA in the Pichia pastoris GS115 successfully. Through classical mouse hot-plate assay and rat hot-tail flick test, the peptide co-CTX MVIIA which expressed in the Pichia pastoris GS115 showed significant analgesic activity.At the same time we developed a useful strategy for preparation of polyclonal antibody against co-CTX MVIIA, and the antibody-based analytical methods for o>CTX MVIIA. It would provide a useful reagent for analysis of co-CTX MVIIA. 1. Development of antibody-based assays for co-CTX MVQ A1.1 Construction of recombinant pGEX-2T-CTX expression plasmidAn artificial DNA sequence encoding co-CTX MWA was designed by using E. co/z-like codons. The plasmid pGEX-2T was digested with BamK VEcoR I and ligated with the annealed product to form the recombinant plasmid pGEX-2T-CTX.1.2 Induced expression of recombinant strain and purification of the fusion protein GST-CTXE coli BL21 transformed with the expression plasmid pGEX-2T-CTX were grown and induced with 0.1 mmol/L IPTG at 37 *C for 3 h. The cells were harvested and lysed by sonication. After centrifugation, the supernatant was applied to Glutathione-Sepharose 4B Fast Flow column. The purified fusion protein showed a single band with molecular weight 28.6 kD on 12 % SDS-PAGE and its purity is about 91.6 % by a computer-assisted scanner. The protein yield was approximately 15 mg/L analyzed with Bradford assay.1.3 Polyclonal antibody preparationTwo adult male rabbits were simultaneously immuned with a mixture of 1.0 mg GST -CTX and the same volume of complete Freund's adjuvant, Every two weeks, 0.5 mg of antigens mixed with an equal volume of incomplete Freund's adjuvant were given subcutaneously for a total of four times.1.4 Polyclonal antibody purificationThe polyclonal antibody was purified by salting out (between 0.3 and 0.5 saturation with ammonium sulfate), and further purified through anion-exchange chromatogra- phy of a DE52 column.1.5 Western blotting to analyse the activity of the polyclonal antibodyThe result of western blotting showed that the antibody of oo-CTX MVDA was able to recognize and bind the two fusion proteins (GST-CTX and Trx-CTX).1.6 Measurement of the titers of the antibody by ELISAGST-CTX and Trx-CTX was coated on the microtiter plate at 4 "C overnight respectively. A normal ELISA system was used to measure the titer of the purified antibody. In terms of the result of ELISA, the titer of the purified antibody against GST-CTX is about 1:65536 and the specific titer of anti-co-CTX MWA antibody was about 1:8192.1.7 Identification of the chemical synthetic version of o-CTX MWADifferent concentrations of the chemical synthetic version of co-CTX MVDA were coated on the microtiter plate. The result of ELISA showed that the value of OD raised as the content of the peptide increased which proved that the antibody-based assays for co-CTX MVEA is feasible. 2. co-conotoxin MVQA expressed in Pichia pastoris has strong bioactivity2.1 Construction of the eukaryotic expression vector of pPIC9-CTXAn artificial DNA sequence encoding co-CTX MWA was designed by using P. pastoris -like codons. The single stranded fragments were annealed to form the paired double strands, the plasmid pPIC9 cut with Xho I / EcoR I was ligated with the annealed product. The recombinant plasmid pPIC9-CTX was subjected to DNA sequencing to confirm the desired gene sequence.2.2 Transformation of Pichia and screening for Mut+ transformantsRecombination plasmid pPIC9-CTX was linearized with Sal I and integrated into P. pastoris GS115 chromsome by electroporation following the standard protocol. Picked one transformant in a regular pattern on both MM and MD plate. After growed 2-4 days at 30 °C, the phenotype of transformants were screened. Mut+ transformantsaccounted for 90 % in total transformants.2.3 PCR analysis of Pichia integrantsUsing genomic DNA isolated from Pichia clones as templates. Amplified the gene of interest by primering with AOXl-up and AOX-l-down. The PCR result showed that the expected size of our interesting gene was about 600 bp. This provided information that the gene of co-CTX MVIIA had been integrated into Pichia genome.2.4 Expression of recombinant Pichia strainSelecting several verified Mut+ transformants at random expressed for 96 h at 28 "C with constant shaking and supplemented with 2.0 % methanol as final concentration every 24 h. After centrifugation, the supernatant was applied to 18 % SDS-PAGE. The result of SDS-PAGE showed that the aimed protein was close-by the molecular weight of 4.1 KDa.2.5 Selecting the high yield recombinant Pichia strainAll P. pastoris clones were inocubated at the same condition to identify the highest yield strain. From the result of SDS-PAGE, it can be observed that the 6th recombinant Pichia strain has the highest level of expression.2.6 Indirect ELISA identificationThe expression supemant of the recombinant Pichia strain was coated on the microtiter plate at 4 TC overnight through covalently linking to the PLL using glutaraldehyde, at the same time the expression supemant of Pichia strain GSl 15 was coated as the negative control. A normal ELISA system was used to identify the peptide co-CTX MWA. In terms of the result of ELISA, the value of OD of the sample was distinctly higher than that of the negative control. It suggested that recombinant co-CTX MVIIA was expressed successfully.2.7 Optimizing the condition of expressionInduction the recombinant Pichia strain with different concentration of methanol, a time course study of expression found the yield of secret protein increased with induced time extended and the concentration of methanol also has crucial effect. After general consideration, we confirmed that the optimized condition was 28 °C ^ 2.0 % methanol and 96 h.2.8 Purification of o-CTX MiAThe supernatant was dialysed overnight using a dialysis membrane with a molecular weight cutoff of 1.0 kDa. The solution was directly applied onto CM-Sepharose Fast Flow. Bound protein was eluted with a linear salt gradient. Fractions containing co-CTX MVII A was pooled and its purity was 90 % evaluated by 18%SDS-PAGE.2.9 Indirect ELISA indentification of the purified w-CTX MVDACoating different concentration of the purified protein on the microtiter plate at 4 "C overnight through covalently linking to the PLL using glutaraldehyde, a normal ELISA system was used to identify the peptide oo-CTX MVDA. In terms of the result of ELISA, the concentration of purified peptide increased, the value of OD raised too. It suggested that the purified peptide was recombinant co-CTX MVILA.2.10 Determination of o>-CTX MIA by MALDI-TOF-MSFrom the result of MALDI-TOF-MS, the molecular weight of recombinant co-CTX MWA expressed by the high yield clone is 2.63889 kDa while natural co-CTX MVEA is 2.63922 kDa. It indicated that 6 sulfhydryl groups of co-CTX MVHA were all oxidized and there was no glycosylation.2.11 Analgesic activity assaysThe marked prolongation in RT was observed in the three groups of dose (PO.01 by two-paired Mext). The analgesic effects (elevated pain thresholds) were dose-dependent. From the data of classical mouse hot-plate method, the threshold that was elevated by recombinant co-CTX MVDA (0.25 ug/kg) is similar with that elevated by morphine (25 ug/kg), so it can be explained that the efficiency to analgesia of recombinant co-CTX MVQA was about 800 times than that of morphine. The similar conclusion can be reached by the rat hot-tail flick test. Altogether, concluding as following:1. Since co-CTX MVflA is a small and disulfide-rich peptide, it is difficult to analy-ze the product. Currently, the chemical synthetic co-CTX MVII A has been analyzed by RP-HPLC, and there are no chemical or immunological assays available for determination of the peptide. Obviously, HPLC can not completely replacespecific chemical and immunological assays for measurement of the conotoxin, especially in unknown samples. Due to its low antigenicity, it is difficult to prepare antibody using co-CTX MVDA as antigen directly. We created a prokaryotic plasmid pGEX-2T-CTX to obtain the highly purified fusion protein GST-CTX, which was directly used to immunize the rabbits to produce the anti-co-CTX MWA antibody. The second fusion protein Trx-CTX was employed to detect the specific part of antibody against the small peptide structure of co-CTX MWA. This strategy is used to produce the polyclonal antibody against small disulfide-rich peptides such as conotoxins. Although it is difficult to obtain enough amounts of conotoxins from the natural or synthetic sources since its low concentration and high expenses, the relatively large amount of the conotoxin can be produced by gene engineering, which solves the problems. As the fusion tags, Trx and GST are not homologous and have no cross reaction, we are able to use Trx-CTX to measure the titer of specific anti-co-CTX MVEA antibody. Our results demonstrated that the antibody of GST-CTX contains the specific anti-co-CTX MWA antibody, which would provide a useful reagent for the analysis of co-CTX MWA.2. From the indirect ELISA and the data of analgesic activity assays, it showed that the new way to produce recombinant co-CTX MWA through eukaryotic expression was successfully and had high biological activity. In this paper, P. pastoris was selected to be the host. As a eukaryote, P. pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being easy to manipulate. It is faster, easier, and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture, and generally gives higher expression level. In summary, co-CTX MWA expressed and purified from P. pastoris expression system possesses significantly analgesic activity. It provides an alternative way to produce large amount of co-CTX MWA for neuroscience research and clinical applications.