The Preparation, Bio-functional Screening And Characterization Of Recombinant Human Pituitary Adenylate Cyclase Activating Polypeptide And Its Derived Peptides

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptidewith multiple important bioactivities and worthy of medical application. In order toproduce non-fusion recombinant human pituitary adenylate cyclase activatingpolypeptide (rhPACAP) using gene engineering technology, a fusion protein with arecognized site of factor Xa (Ile-Glu-Gly-Arg↓) between CBD (cellulose bindingdomain) and rhPACAP was expressed and purified by cellulose affinitychromatography. Non-fusion rhPACAP was released by the cleavage of factor Xa,then purified by HPLC and identified by Western blot and Laser flying massspectrometry. The preliminary bioactivity assay indicated that the product had theactivity of promoting cAmp in the cell line SW1990 of human pancreas carcinoma.A short flexible peptide abundant in Gly(Gly-Thr-Gly-Gly-Gly-Ser-Gly) wasadded before the recognized site by factor Xa. The compare of the cleavage efficiencyof factor Xa on both two-group fusion proteins (CBD-IGF and CBD-PACAP) withand without short flexible peptide indicated that the short peptide helped to improvethe sensitivity of fusion protein to factor Xa in different degree. A strategy ofpromoting the cleavage efficiency of factor Xa was presented.In order to obtain the recombinant human PACAP efficiently by intein-mediatedsingle column purification, the target protein was over-expressed as a fusion to theN-terminus of a self-cleavable affinity tag. After the PACAP-intein-CBD fusionprotein was purified by chitin-affinity chromatography, the self-cleavage activity ofthe intein was induced by thiol and the RMPACAP was released from thechitin-bound intein tag. The activity assay showed that RMPACP, which has an extraMet at its N-terminus compared with the native human PACAP, had the similaractivity of stimulating cAmp accumulation with the standard PACAP38 in theSW1990 cells. Twenty-two milligrams of RMPACAP with the purity over 98% was obtained by single column purification from 1 liter of induced culture. A new efficientproduction procedure of the active recombinant human PACAP was established.Four recombinant PACAP derived peptides (RMROM, RROM, RMBAY andRBAY) as potential therapies for Typeâ…¡Diabetes were designed based on theknown agonist of VPAC2 receptor, and were prepared using intein-mediatedpurification system. All peptides (50ng/kg) decreased the level of plasma glucose afterinfused with high concentration glucose (1.8mmol/kg) to the enterocoelia of NIHmouse. RMBAY with the highest activity of reducing the level of plasma glucosestimulated the glucose-dependent insulin secretion. The screening and preparation ofRMBAY laid the foundation for development of RMBAY as potential therapy forType 2 Diabetes.The receptor-transfected Chinese hamster ovary (CHO) cell clones expressingthree receptors subclasses of PACAP respectively were constructed and selected. Theresults of RT-PCR, immunoflourescence assay and the binding assay of¹²⁵I-PACAP38 to the receptor showed that three receptors were expressed on threecells (VPAC1-CHO, VPAC2-CHO and PAC1-CHO) respectively. The competitionbinding of ¹²⁵I-PACAP38 on membranes from CHO cells expressing each of threesubtypes of human PACAP receptors was used to identify the selectivity of RMBAYand RMPACAP on three receptors. RMPACAP competitively displaced¹²⁵I-PACAP38 on all three receptor subclasses. RMBAY competitively displaced¹²⁵I-PACAP38 on VPAC2 with a half-maximal inhibitory concentration (IC50) of 70±5nmol/L, whereas the IC50 of RMBAY at human VPAC1 or PAC1 was observedup to 10μmol/L1. RMBAY stimulated the cAmp in VPAC2-CHO cells with ahalf-maximal stimulatory concentration (EC50) of 1.8±0.2nmol/L. Thus, RMBAYis a selective agonist ligand for VPAC2 receptor subclass.The intein-mediated purification and cyclization of unprotected peptidethioesters were tried to produce a recombinant cyclic peptide (RCBAY) for thepurpose of improving the stability and activity of the peptide. The mass spectrometryassay showed that the products contained the component with the molecular weight of4128.10 consistent with the theoretic value of RCBAY. The mixture containing RCBAY displayed a stronger effect on reducing the level of plasma glucose and lastedlonger time than RMBAY. It was suggested that RCBAY might be a more effectivetherapy for typeâ…¡diabetes.Some parameters were studied, e.g. the growth curve of the engineered strainRMBAY-ER2566, the value of OD₆₀₀ at which the expression of the target protein wasinduced, the amount of IPTG and the time spent on inducing protein expression. Thetarget protein was effectively induced by 0.4mmol/L IPTG for 3-4hr at 30℃or for2-3hr at 37℃, when the OD₆₀₀ of the culture reached 0.5-1. Glucose inhibited theexpression of the target fusion protein. Based on the conditions mentioned above, thestrain was fermented with high density in 5L fermentor. One hundred and thirteengrams of wet bacteria was obtained from 3.5L culture. The expression of target fusionprotein reached 32-35% of total bacterial protein and 28% of the total soluble proteins.The fermentation of the engineered strain RMBAY-ER2566 ensured the furtherresearch and medical development of RMBAY.All in a word, the study focused on the efficient preparation of recombinantPACAP and its derived peptides, the screening and identification of RMBAY as anovel agonist of VPAC2 receptor with bioactivity of reducing plasma glucose,producing cyclopeptide RCBAY based on RMBAY for improving the bioactivity andstability of the peptide, and the fermentation of the engineered strain for the furtherresearch and application of the novel agonist of VPAC2 receptor of which we ownedthe property right.