Study On Expression Of Recombinational Antimicrobial Peptide Mediated By Inclusion Body

Antimicrobial peptide?AMP?is a kind of small molecule cationic peptide,it has immunoregulation effect, sterilization effect and so on. Inclusion body?IB? is a protein aggreations which recombinant proteins over expresssed at Escherichia coli. Meanwhile, it is one form of recombiant proteins expressed at Escherichia coli.Using fusion tags which are apt to form inclusion body express with antimicrobial peptides not only can improve the content of fusion proteins, but also enhance its stability.However, fusion tags must be removed soon afterwards. At present, there are several ways removing fusion tags. However, those are some toxic chemicals, for example Bromide nitrile,Hydroxylamine or Formic acid. But the cost of depuration by using High performance liquid chromatography?HPLC? is too high. Fusion tags can be separated from recombinant proteins through Ni²⁺'s effectively cleavage at a specific loci. At the same time, Ni²⁺can be chelated by EDTA, and chelation can be removed though purification at the next step.This research takes Mehchnikowin and Maginin ? as antimicrobial peptide. Selecting proteins which easy to form inclusion body, Green fluorescent protein?GFP?,Palmitoyl phospholipids transferase?Pag P? and Group of protein folding structure domain?TAF? as fusion tags to build a series of exprssion fusion proteins(insert recognition sites between fusion tag and antimicrobial peptides where catalyzed cleavage site by Ni²⁺, His6-tag depuration label and recognition site of TEV protease) and relevant expression vectors.Making use of Escherichia coli expression system to obtain inclusion bodies, and through SDS-PAGE, analysis of gray and Western Blotting proving. Finally, this reseach screen out Pag P which expresses comparatively higher, also figure out the best time? 10 h? and concentration of IPTG?0.5 m M?. Dissolving the inclusions in Hepes buffer?p H8.2? with 6 M guanidine hydrochloride, then using Ni2 +catalyzed cleavage. In order to reseach the bestenvironment of Ni²⁺catalyzed cleavage, this study designed three factors include response-times, temperatures and fusion-protein's concentrationes. Results show that under the condition 60?, 200 ?M fusion protein, after reacting 24 h. Ni2 +can effectively cleavage fusion protein, forming fusion tag Pag P and short peptide HT-AMPs. Fusion tags what were catalyzed-cleavaged by Ni²⁺could wiped off by diluting precipitation with the corresponding buffer. HT-AMPs can be received a further purification through Ni-NTA. Using TEV peotease to digest the purified product, then obtain the pure of antimicrobial peptide.This research also measured the activity of antimicrobial peptide. Bacteriostasis experiments shows, although HT-Mag ??HT-Metch? has been digestion by TEV protease, but it has no biological activity. Without desalting, all can have showed bacteriostatic activity.Mag ? can kill Escherichia coli MG 1655 and Staphylococcus Aureus, show of gram positive and negative bacteria's micrococcus. Metch can kill the Bacillus subtilis and Micrococcus luteus, showed gram-positive bacteria micrococcus.