K Seetha RAM, M Mary Vijaya KUMAR, S Divya SRİ, N Bhargava RAMUDU, J B PERAVALİ, Kk PULİCHERLA

Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158

Amaç: Sentetik katyonik antimikrobiyal peptid (SK-AMP) önemi gittikçe artan ve konvansiyonel antibiyotiklere alternatif olması beklenen terapötik bir moleküldür. Bu çalışmada SK-AMP’yi intein tabanlı bir sistem ile enzimatik parçalanma olmaksızın saflaştırmak amaçlanmıştır.Yöntemler: İntein sekansı pTYB11 vektöründen PZR kullanılarak amplifiye edildi ve inteinin N-terminal ucu SK-AMP ile bağlandı. Oluşturulan bu yapı soğuk şok ekspresyon vektörü MCS bölgesine klonlandı. pCOLDI ve rekombinan peptid kitin afinite kodonundan 50 mM DDT ile ayrılarak enzimatik ayrıştırma olmaksızın toplandı. Daha sonra toplanan peptitin miktarı belirlendi ve saflaştırılan pepitd yapının antimikrobiyal aktivitesi difüzyon yöntemi ile belirlendi.Bulgular: Başlangıçta intein bağlı SK-AMP, IPTG indüklenebilir E. coli BL21(DE3) ve tuz indüklenebilir E. coli GJ1158’de bir füzyon protein olarak eksprese edildi. Tuz ile indüklenen E. coli GJ1158’de kitin bağlayan kodonun kullanıldığı tek basamaklı saflaştırma ile 208 mg/L konsantrasyonunda çözünebilir SK-AMP elde edildi. Saflaştırılmış SK-AMP’nin antibakteriyel etkisi hem gram pozitif hem de gram negatif bakterilere karşı gösterildi ve minimum inhibitör konsantrasyonu belirlendi.Sonuçlar: İlk kez çözünebilir SK-AMP tuz ile indüklenebilir E. coli GJ1158’den kitin bağlayan kodon kullanılarak tek basamakta enzimatik ayrışma olmaksızın elde edildi

Anahtar Kelimeler:

Sentetik katyonik antimikrobiyal peptid, intein sekansı, pCOLDI, kitin bağlayan kodon, E. coli BL21

Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158

Objective: Synthetic cationic antimicrobial peptide (SC-AMP) is an important and upcoming therapeutic molecule against conventional antibiotics. In this study, an attempt was made to purify the SC-AMP without the enzymatic cleavage of the affinity tag, by using an intein-based system. Methods: The intein sequence was amplified from pTYB11 vector using PCR methodologies and the N-terminal of intein was ligated with SC-AMP. The designed construct, intein-SC-AMP was cloned into MCS region of cold shock expression vector, pCOLDI and the recombinant peptide was purified on a chitin affinity column by cleaving intein with 50 mM DTT without applying enzymatic cleavage. Later the peptide was quantified and its antibacterial activity of the purified peptide was studied using well diffusion method. Results: Initially, intein-SC-AMP was expressed as a fusion protein in both IPTG inducible E. coli BL21(DE3) and salt inducible E. coli GJ1158. Single step purification using CBD (chitin binding domain) - intein tag in salt inducible E. coli GJ1158, yields the SC-AMP in the soluble form at a concentration of 208 mg/L. The antibacterial activity and minimal inhibitory concentration (MIC) of the purified SC-AMP was studied against both Gram positive and Gram negative microorganisms. Conclusion: For the first time, single step purification of soluble SC-AMP was carried out using chitin-binding domain affinity tag in salt inducible E. coli GJ1158 without an application of enzymatic cleavage.

Keywords:

Synthetic cationic antimicrobial peptide, intein sequence, pCOLDI, chitin affinity column, E. coli BL21(DE3),

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