Safety Aspect of Recombinant Protein Produced by Escherichia coli: Toxin Evaluation with Strain and Genomic Approach

Enterokinase is a serine protease commonly used in some biotechnology researches. For these purposes, the light chain containing enterokinase activity has usually been expressed as recombinant protein in different expression systems because natural enterokinase extraction is often ineffective. In this study, we examined the formation of recombinant enterokinase expressed in Escherichia coli with biological activity. The thioredoxin-enterokinase (trx-ent) fusion protein was autocleavaged into thioredoxin and enterokinase when expressed under insoluble form, denatured with guanidine and then refolded with suitable oxidation and reduction steps. Meanwhile, soluble expression as well as insoluble form denatured by urea had not enzymatic activity. Denaturant solution of 6 M guanidine along with the re-folding conditions in oxidized glutathione oxidation buffers followed by the reduced glutathione buffer with arginine was applied to produce trx-ent protein capable of self-cleavage. The recombinant light-chain enterokinase protein had a size of about 35 kDa on the Tris-glycine gel. Initial assessment on substance had shown that enterokinase was capable of cleaving thioredoxin-sumoprotease into thioredoxin and sumoprotease. This result provides the base for the production of active recombinant enterokinase to be used in recombinant protein expression technology.

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Cloning and expression of Human Papilloma virus type 16 L1 capsid protein in bacteria

Health Science Journal of Indonesia ◽

10.22435/hsji.v12i2.2442 ◽

2019 ◽

Vol 10 (2) ◽

pp. 82-89

Author(s):

Silvia Tri Widyaningtyas ◽

Sofy Meilany ◽

Budiman Bela

Keyword(s):

Escherichia Coli ◽

Cervical Cancer ◽

Human Papillomavirus ◽

Recombinant Protein ◽

Capsid Protein ◽

Recombinant Plasmid ◽

Health Science ◽

Science Journal ◽

Hpv 16 ◽

L1 Protein

Latar belakang: Secara alamiah protein kapsid L1 Human Papillomavirus (HPV) tipe 16 dapat mengalami auto assembly untuk membentuk Viral like particle (VLP). Terkait dengan penelitian vaksin HPV, VLP dapat digunakan untuk berbagai keperluan seperti vaksin, pseudovirion atau SpyTag-Spycatcher. Penelitian ini ditujukan untuk mendapatkan plasmid rekombinan yang digunakan untuk produksi protein L1 HPV 16. Metode: Gen penyandi protein L1 HPV 16 diklona ke dalam vector pQE80L, suatu plasmid yang mengandung sistem ekspresi untuk prokariota. DNA penyandi HPV 16 L1 disisipkan pada situs restriksi BamHI dan Hind III plasmid pQE80L. Plasmid rekombinan yang mengandung gen L1 HPV 16dikonfirmasi menggunakan PCR dan analisis enzim restriksi. Lebih lanjut untuk memastikan bahwa gen rekombinan L1 HPV 16 dapat diekspresikan dalam prokariota, plasmid rekombinan ditransformasikan ke bakteri Escherichia coli BL21 (DE3). Bakteri diinduksi dengan Isopropyl β-D-1-thiogalactopyranoside (IPTG) dengan berbagai konsentrasi dan berbagai waktu inkubasi. Hasil: protein rekombinan L1, berat 56 kDa, telah berhasil diekspresikan dalam sistem prokariota. Protein rekombinan L1 dapat dimurnikan menggunakan TalonR dalam kondisi denaturasi. Kesimpulan: gen L1 HPV 16 telah dikloning ke dalam pQE80L dan berhasil diekspresikan dalam sistem prokariota. (Health Science Journal of Indonesia 2019;10(2):82-9) Kata kunci: L1, HPV 16, cervical cancer Abstract Background: Naturally Human Papillomavirus (HPV) type 16 L1 capsid protein can auto assemble to form Viral like particles (VLP). Concerning to vaccine development for HPV, VLP can be used for a variety of needs such as a vaccine, pseudovirion or SpyTag-Spycatcher. In this study, to obtain a vector expression that can be used in the production of HPV L1 protein, we cloned gene coding HPV 16 L1 protein into pQE80L a plasmid contains an expression system for prokaryote. Methods: The DNA coding HPV 16 L1 was inserted at BamHI and Hind III restriction sites of pQE80L plasmid. The recombinant plasmid containing the HPV L1 gene was confirmed using PCR colony and enzyme restriction. Further to ensure the recombinant HPV 16 L1 gene could be expressed in a prokaryote, the recombinant plasmid was transformed into bacteria Escherichia coli BL21 (DE3). The bacteria were induced with IPTG with various concentrations and various incubation time. Result: L1 recombinant protein, 56 kDa in weight, has successfully been expressed in prokaryote system. L1 recombinant protein can be purified using TalonR under denaturing conditions. Conclusion: L1 HPV 16 gene has been cloned into pQE80L and successfully expressed in prokaryote system. (Health Science Journal of Indonesia 2019;10(2):82-9) Keywords: L1, HPV 16, cervical cancer

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