Construction of an Expression System for Aqualysin I in<i>Escherichia coli</i>That Gives a Markedly Improved Yield of the Enzyme Protein

Sakaguchi, Minoru; Niimiya, Keisuke; Takezawa, Makoto; Toki, Tsutomu; Sugahara, Yoshiyuki; Kawakita, Masao

doi:10.1271/bbb.80132

Cited by 7 publications

(4 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To overcome this problem, we dialysed the fusion protein cleavage mixture against 20 mM Tris/HCl, 25 mM NaCl, 0.5 M urea and 0.05% SDS (pH 9.5) at 4 °C. This method is known to increase the solubility of HBx protein while avoiding denaturation processes [25]. After dialysis, the solution appeared clear.…”

Section: Resultsmentioning

confidence: 99%

High‐level expression and large‐scale preparation of soluble HBx antigen from Escherichia coli

Liu

Zou

et al. 2009

Biotech and App Biochem

View full text Add to dashboard Cite

The HBx (hepatitis B virus X protein) is a multifunctional regulator of cellular signal transduction and transcription pathways in host-infected cells. Evidence suggests that HBx has a critical role in the pathogenesis of hepatocellular carcinoma. However, the lack of efficient large-scale preparation methods for soluble HBx has hindered studies on the structure and function of HBx. Here, a new pMAL-c2x protein fusion and purification system was used for high-level expression of soluble HBx fusion protein. The high-purity fusion protein was obtained via amylose resin chromatography and Q-Sepharose chromatography. The untagged HBx was efficiently and rapidly purified by Sephadex G-75 chromatography after cleavage by Factor Xa at 23 °C. The purity of active HBx protein was >99% with a very stable secondary structure dominated by α-helix, β-sheet and random structure. The purified HBx protein can be analysed to determine its crystal structure and function and its capabilities as an effective immunogen.

show abstract

Section: Resultsmentioning

confidence: 99%

High‐level expression and large‐scale preparation of soluble HBx antigen from Escherichia coli

Liu

Zou

et al. 2009

Biotech and App Biochem

View full text Add to dashboard Cite

show abstract

“…The plasmid pMAQΔc2, which was designed to express wild-type AQN as a fusion protein with maltose binding protein (MBP), was constructed based on pAQNΔC105 and pMAL plasmids (New England Biolabs, Ipswich, MA, USA) as described previously (Sakaguchi et al [2008a]).…”

Section: Methodsmentioning

confidence: 99%

“…The cells were harvested by centrifugation and subsequently sonicated, and the crude extract was subjected to heat treatment, hydrophobic chromatography (Butyl Sepharose; GE Healthcare, Buckinghamshire, UK) and cation exchange chromatography (Resource S; GE Healthcare) as described previously (Sakaguchi et al [2008a]). The enzymes were purified to homogeneity to yield a single band on SDS-polyacrylamide gel electrophoresis (PAGE) after staining with Coomassie Brilliant Blue R-250 (CBB R-250) (Laemmli [1970]).…”

Section: Methodsmentioning

confidence: 99%

Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1

et al. 2014

Self Cite

View full text Add to dashboard Cite

The proteinase K subfamily enzymes, thermophilic Aqualysin I (AQN) from Thermus aquaticus YT-1 and psychrophilic serine protease (VPR) from Vibrio sp. PA-44, have six and seven salt bridges, respectively. To understand the possible significance of salt bridges in the thermal stability of AQN, we prepared mutant proteins in which amino acid residues participating in salt bridges common to proteinase K subfamily members and intrinsic to AQN were replaced to disrupt the bridges one at a time. Disruption of a salt bridge common to proteinase K subfamily enzymes in the D183N mutant resulted in a significant reduction in thermal stability, and a massive change in the content of the secondary structure was observed, even at 70°C, in the circular dichroism (CD) analysis. These results indicate that the common salt bridge Asp183-Arg12 is important in maintaining the conformation of proteinase K subfamily enzymes and suggest the importance of proximity between the regions around Asp183 and the N-terminal region around Arg12. Of the three mutants that lack an AQN intrinsic salt bridge, D212N was more prone to unfolding at 80°C than the wild-type enzyme. Similarly, D17N and E237Q were less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis. The AQN intrinsic salt bridges appear to confer additional thermal stability to this enzyme. These findings will further our understanding of the factors involved in stabilizing protein structure.

show abstract

“…For scientific and industrial purposes, different systems have been used for cloning and expression of xylanolytic enzymes, especially from fungi. Recombinant Escherichia coli, yeast and fungi have been studied, and each system has advantages and disadvantages that must be evaluated in order to obtain functional recombinant enzyme (Caoet al 2007;Carapito et al 2009;Nevalainen et al 2005;Sakaguchi et al 2008). Among these systems, E. coli has been widely used for the cloning and heterologous expression of recombinant xylanases from fungi Lee et al 2007).…”

Section: Regiões Intragênicas No Gene Pwl2mentioning

confidence: 99%

Expressão, purificação e caracterização parcial de proteínas relacionadas à patogenicidade de Magnaporthe grisea

Schneider¹

View full text Add to dashboard Cite

Para meus pais, Dimas e Eclair, que me presentearam com o seu imenso e acalentador amor altruísta.v AgradecimentosTudo o que sou, de algum modo, procede de alguém. Assim, agradeço:Ao único Deus verdadeiro, pela dádiva preciosa da vida possibilitando-me perceber e analisar a sua grandiosa criação; Aos meus pais, meus irmãos e minhas irmãs que sempre me asseguraram de sua amizade incondicional -em especial ao Dilson, pelo encorajamento e pela companhia; Aos meus amados filhos (Elisa, Tomás, Hugo e Álvaro), pelo amparo de seu terno amor e porque, apesar de jovens, souberam aguardar pacientemente o "fim da tese da mãe"; Ao Homero, marido e companheiro, pelo apoio e pela compreensão; À Anete, que, além de excelente orientadora neste trabalho e referência profissional de liderança, não se esquivou de me apoiar em assuntos pessoais. Você será sempre uma grata lembrança para mim, Anete! Aos meus colegas e amigos do LAGM/CBMEG/UNICAMP: Alexandre, Carol, Clelton, Eliane, Karen, Lilian, Letícia, Luciana, Marianna, Roberta e Susy pelo conhecimento compartilhado, pelo auxílio nos experimentos e pelo ambiente de trabalho que cada um proporcionou dentro de sua capacidade; em especial, ao Adriano, ao Antonio, à Camília, à Cleide, ao Marcelo e ao Zé Sérgio que também me permitiram dividir, com eles, anseios e dificuldades pessoais; Aos meus colegas e amigos do Barracão da Genética/UNICAMP pelo auxílio profissional e pelos momentos agradáveis (como nas festinhas e nos churrascos);Ao Juverlande e à Rosana: meus sempre grandes amigos; À Zilda, com seu sorriso fácil e sua boa vontade, por ter providenciado prontamente o material de laboratório adequadamente limpo e esterilizado e pelo seu café sempre revigorante; À Tânia, à Sandra e à Lourdes pela eficiência no cumprimento de suas funções; Aos membros da pré-banca e da banca de defesa de tese por sua pronta disposição em utilizar seus conhecimentos no julgamento desta tese; vi À CAPES e à FAPESP pelo apoio financeiro e pelas avaliações dos relatórios; À UNICAMP, por oferecer a estrutura física e a estrutura administrativa adequadas para o desenvolvimento deste trabalho.

show abstract

Construction of an Expression System for Aqualysin I inEscherichia coliThat Gives a Markedly Improved Yield of the Enzyme Protein

Cited by 7 publications

References 15 publications

High‐level expression and large‐scale preparation of soluble HBx antigen from Escherichia coli

High‐level expression and large‐scale preparation of soluble HBx antigen from Escherichia coli

Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1

Expressão, purificação e caracterização parcial de proteínas relacionadas à patogenicidade de Magnaporthe grisea

Contact Info

Product

Resources

About