[논문]Production of a Fusion Protein Containing the Antigenic Domain 1 of Human Cytomegalovirus Glycoprotein B

Sousa Fani; Ferreira Susana; Queiroz Joao; Domingues Fernanda

Production of a Fusion Protein Containing the Antigenic Domain 1 of Human Cytomegalovirus Glycoprotein B 원문보기

Journal of microbiology and biotechnology, v.16 no.7, 2006년, pp.1026 - 1031

Sousa Fani (Centro de Investigacao em Ciencias da Saude, Universidade da Beira Interior) , Ferreira Susana (Centro de Investigacao em Ciencias da Saude, Universidade da Beira Interior) , Queiroz Joao (Centro de Investigacao em Ciencias da Saude, Universidade da Beira Interior) , Domingues Fernanda (Centro de Investigacao em Ciencias da Saude, Universidade da Beira Interior)

Abstract ▼ AI-Helper

The optimization of the production of a fusion protein containing the antigenic domain 1 (AD-1) is of a great importance, considering its use in diagnostic tests. The fusion protein is produced by the fermentation of a recombinant strain of Escherichia coli containing the plasmid Mbg58, which expresses the AD-1 (aa 484-650) of human cytomegalovirus glycoprotein B as a fusion protein together with aa 1-375 of ${\beta}-galactosidase$. An important characteristic of promoters (lac and derivatives) used in recombinant protein production in E. coli is their inducibility. Induction by IPTG is widely used for basic research; however, its use in large-scale production is undesirable because of its high cost and toxicity. In this work, studies using different inducers and carbon sources for the production of a fusion protein containing the AD-l were performed. The results showed that lactose could be used as an inducer in the fermentation process for the production of this protein, and that expression levels could exceed those achieved with IPTG. The use of lactose for protein expression in E. coli should be extremely useful for the inexpensive, large-scale production of heterologous proteins in E. coli. Addition of sucrose to the fermentation medium improved the yield of recombinant protein, whereas addition of fructose or trehalose decreased the yield.

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가설 설정

A. Cell growth curves obtained by addition of different disaccharides to the medium: without di saccharides (O); with 100 mM sucrose (*); with 100 mM trehalose (▲). B.

제안 방법

5, with the aim of avoiding inhibitory effects that could eventually occur because of a high residual concentration of lactose in the medium. The best results for fusion protein production were achieved at OD600 value of 1; the cells were induced at that OD600 value in all the experiments performed in this work.

이론/모형

A 30-min gel staining with Coomassie Brilliant Blue R-250 solution was performed in order to visualize fusion protein as a major band close to the 66 kDa protein marker. Quantitative measurements of p-galactosidase activity in liquid cultures were done using the P-galactosidase reporter gene activity detection kit (Sigma), based on Miller's method. Cell lysis was carried out by toluene/ deoxycholate solution.

성능/효과

The results of P-galactosidase activity measured throughout the induction phase showed nearly undetectable levels even 4 h after induction, confirming that repression occurred when the cells were grown on 100 mM fructose or glucose. The addition of 1.

참고문헌 (19)

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Production of a Fusion Protein Containing the Antigenic Domain 1 of Human Cytomegalovirus Glycoprotein B 원문보기

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Production of a Fusion Protein Containing the Antigenic Domain 1 of Human Cytomegalovirus Glycoprotein B 원문보기

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