Part:BBa_K3098025
DSAOP+Avi-tag
Free radicals, such as superoxide anions (O2·-) or hydroxyl radicals (HO·), can cause protein damage and DNA mutations that can initiate several diseases, including coronary heart diseases and diabetes. Antioxidants that can scavenge free radicals play an important role in biological systems.An antioxidant peptide derived from Pinctada fucata has great poteintial for scavenging free radicals. Howeverthe the low rates of enzymatic hydrolysis extraction and purification, as well as the higher costs for purifying from Pinctada fucata meat, suggest that this natural and safe antioxidant peptide would not meet industrialized production requirements. Thus we have submited a designed AOP that can be easily produced and purified in E.coli, named 'DSAOP' BBa_K3098024. For our project need to assemble this DSAOP onto the Bacteria cellulose via a connection of streptomycin and biotin. Thus we add Avitag onto the N terminal of DSAOP which can link with biotin under the catalyzation of the enzyme BirA K2485001.So that DSAOP can be biotinated during its production in E.coli.
Usage and Biology
The sequence of antioxidant peptides isolated from Pinctada fucata meat typically includes 10 to 20 amino acids, which is relatively short and easily degraded by protease or peptidase in Escherichia coli expression systems. Therefore, it is difficult to express these peptides directly. Based on previous reports that an antioxidant peptide from Pinctada fucata meat (amino acid sequence was GAGLPGKRER, named AOP) had high antioxidant activity in vitro, a design containing the GAGLPGKRER sequence was constructed for further expression to obtain a higher yield and improve its stability in our project’s applications in room temperature environment. Meanwhile,and solubility of the designed polypeptide were considered.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 102
Illegal BamHI site found at 145
Illegal XhoI site found at 343 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Results
| chassis | E.coli BL21 |
1.Measurement of DPPH Radical Scavenging Activity
The DSAOP antioxidant activity was determined using the DPPH radical scavenging assay. DPPH, also known as 1, 1-diphenyl-2-trinitrophenylhydrazine, is a very stable free radical in the nitrogen center. Its stability mainly comes from the space barrier of the three benzene rings with resonance stabilization effect, so that the unpaired electrons on the middle nitrogen atoms cannot play their due electron pairing effect. Its size of anhydrous ethanol was purple in color, with maximum absorption at 517nm wavelength and linear relationship between absorbance and concentration. When free radical scavenger is added to it, DPPH can be combined or replaced to reduce the number of free radicals, reduce the absorbance, and make the solution color become lighter, so as to evaluate the ability of scavenging free radicals. In other words, the antioxidant capacity was calculated by detecting the effect of removing DPPH from the sample at the wavelength of 51 7nm.The DPPH radical scavenging activity was calculated as follows: DPPH radical scavenging activity (100%) = [1 − (Ai − Aj )/ A0] × 100.
The following is the result of the DPPH radical scavenging activity of DSAOP compared with DSAOP+Avitag and Vitamin E. The DSAOP has the best scavenging activity and enduring ability.
2.Measurement of Hydroxyl radical scavenging activity
Fenton reaction to generate hydroxyl free radicals react with salicylic acid, generated at 510 nm with special absorption of 2, 3 - dihydroxy benzoic acid, reactive is as follows: if to join in reaction system which has the function of removing hydroxyl free radical was tested, will reduce the number of generated hydroxyl free radicals, thus reduce the generation of coloured compounds corresponding. Fixed reaction time was used Method, The absorbance of the reaction solution containing the measured substance was measured at 510 nm and compared with the blank solution to determine the hydroxyl content of the measured substance Clearance of bases.
The following picture is the result of the Hydroxyl radical scavenging activity compared with DSAOP+Avitag and Vitamin E. The DSAOP - Avitag has the best hydroxyl scavenging activity and the next one is DSAOP.
| None |