Preparation And Functional Study Of Recombinant Protein PTD-MAX With Transmembrane Activity

PAC1 was the specific receptor of pituitary adenylate cyclase activating polypeptide(PACAP) with activating important biological functions in organisms. Maxadilan (nerve-polypeptides) extracted from the sand fly salivary gland was a specific agonist for the PAC1 performed biological functions during nerve-injury remedy or energy metabolism regulation. PTD (Protein transduction domain) was a domain of TAT protein with effective transmembrane ability, which effectively conducts the linked large molecule protein into membrane. In this research, the recombinant PTD-MAX consisting of PTD and Maxadilan was prepared efficiently by using genetic engineering principle and technology. The transmembrane ability, biological activity and drug applications of the recombinant PTD-MAX were also studied. These experimental results for recombinant PTD-MAX will lay the foundation on the development of potent therapy drug with transmembran activity for the cornea bioremediation and drug application in brain, eye and other neural-intensive organisms.The engineering bacteria strain pKYB-PTD-MAX-ER2566 was constructed based on the already existing PTD in Lab and well known Maxadilan sequence by overlap PCR technique.The conditions for engineering bacteria fermentation in shake flask and 100L bioreactor were investigated. The fermentation conditions for the recombinant PTD-MAX optimum expression were controlled at 2% of glycerol as a carbon source, incubated for 2-3 hours at 37℃, added IPTG(isopropyl P-D-thiogalactoside) with 0.5mmol/L of the concentration, induced cultivation for 3-4 hours until OD600 reached at 0.5-1.0, and 30.4g/L of bacteria culture were obtained from 100L bioreactor cultivation process.12.1 mg/L recombinant PTD-MAX with the purity over 95% were obtained by use of Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) protein purification system and affinity chromatography.The transmembrane ability of the recombinant protein PTD-MAX to CHO cell membrane was investigated according to FITC (fluorescein isothiocyanate) technique. The experimental results revealed that PTD-MAX displayed the transmembrane activity, not only effectively pass through the CHO cell membrane, but also permeated mouse blood-brain barrier and penetrating through eye cornea of mice and rabbit. The efficiency of recombinant PTD-MAX permeating the mice's blood-brain barrier was significantly higher than that of Maxadilan (P<0.01). The efficiency of recombinant PTD-MAX penetrating through eye cornea of mice and rabbit was 4.7 times and 7.2 times respectively than that of Maxadilan.The study results on the biological activity of recombinant protein PTD-MAX showed that the recombinant PTD-MAX could increase plasma glucose instantly and inhibited the food intake more significantly for tested mice than Maxadilan. These results indicated that PTD-MAX preserved the biological activity of natural Maxadilan and activated the PAC1 effectively.The experimental results on the cornea repair of recombinant protein PTD-MAX for rabbit's corneal alkali injury indicated that PTD-MAX promoted effectively the healing of corneal ulcers, reduced the Inflammation reactions and improved the sensitivity of the cornea compared with the control group. These results suggested that recombinant PTD-MAX had a therapeutic effect on cornea injury.