Cloning, Expression, Purification And Bioactivities Of PACAP And Its Derivative

Pituitary adenylate cyclase-activating polypeptide(PACAP) shares 68% identity at the amino acid level with vasoactive intestinal polypeptide (VIP), and belongs to VIP/secretin/glucagon/growth hormone-releasing hormone (GHRH) super-family. It's a pleiotropic polypeptide which can protect humans from diseases, such as Diabetes, Obesity, Asthma, Parkinson, etc, to some extent. However, PACAP has a short half-life that less than 10min in vivo. What's more, there is no better method to prolong it except chemical modification in vitro until now. Here, a new derivative that can be prepared totally using genetic engineering technology is studied. With one Cysteine added on the C-terminus, it is possible for the derivative to bind serum albumin in vivo, thus the stability of the peptide may be improved.The study comprises three parts as follows:1) Cloning, expression and purification for PACAP and its derivativeWe modified the GenPept sequence of PACAP (PACAP38 and PACAP27) and named the derivative cPACAP (cPACAP38 and cPACAP27). The gene sequences of PACAP38, PACAP27 and cPACAP27 were obtained via PCR with synthesized cPACAP38-gene as template. Then cloned these four genes into pET32a(+)/BL21 (DE3) seperately, so that peptide could be fused with thioredoxin (Trx).After inducing the four engineered strains described above by IPTG, four fusion proteins were obtained, and the expression and solubility percentages of these four fusion proteins were analyzed by ImageJ. Results showed that the percentages were 22.1% and 48.7% for Trx-PACAP38,24.6% and 54.0% for Trx-cPACAP38,47.3% and 88.3% for Trx-PACAP27,47.7% and 66.6% for Trx-cPACAP27.The induced bacteria was sonicated, and then purified by twice Ni2+ affinity chromatography. Finally,1.6mg PACAP38 with 95% purity,1.8mg cPACAP38 with 98% purity,9.1mg PACAP27 with 99% purity and 5.7mg cPACAP27 with 99% purity were prepared for further study. 2) Bioactivity validation and comparison of PACAP and its derivativeTo validate and compare the bioactivity of the engineered PACAP and its derivative, we tested the cumulative food intake of mice that were intraperitoneally administered with PACAP or cPACAP. Results showed that both PACAP and cPACAP displayed anorexigenic activity to mice, but cPACAP, including cPACAP38 and cPACAP27, was much more effective on inhibition ratio and time than PACAP, indicating that cPACAP was more stable and has longer half-life than PACAP.3) Bioactivities study of PACAP and its derivativeTo research whether PACAP or cPACAP can cure obesity, mice were treated with PACAP38 or cPACAP38 for 10 days. During the administration, body weights of mice were tracked. On the 11th day, intraperitoneal glucose tolerance, the serum triglyceride, high density lipoprotein, low density lipoprotein and cholesterol were measured. Results showed no difference between treated and untreated mice.To test the regulatory effect of PACAP and cPACAP on the proliferation of tumor cells, four human cancer cell lines, breast cancer cell Bcap37, hepatic cancer cell BEL7404, neuroblastoma cell SH-SY5Y and lung cancer cell A549, were analy-zed by MTT after treated by PACAP27 or cPACAP27. No difference again.Then PACAP27 or cPACAP27 was intraperitoneally injected to mice together with GLP-1, to study the effect of PACAP and cPACAP on the bioactivity of GLP-1. The plasma glucose data of treated mice indicated that PACAP and cPACAP help GLP-1 to decrease plasma glucose, because the decreasing effect was 3 times longer when PACAP or cPACAP worked together with GLP-1 than that when treated by GLP-1 alone.Briefly, we got four genetic engineering strains and validated that this new PACAP derivative with one Cysteine on the C-terminus indeed displayed prolonged activity in vivo. What's more, we proved that PACAP or cPACAP has positive effect on the hypoglycemic effect of GLP-1, laying a foundation for further study or utility.