A simple strategy for the purification of native recombinant full-length human RPL10 protein from inclusion bodies

Pereira, Larissa Miranda; Silva, Luana R.; Alves, Joseane F.; Marin, Nélida; Silva, Flavio Sousa; Morganti, Lígia; Silva, Ismael D.C.G.; Affonso, Regina

doi:10.1016/j.pep.2014.06.003

Cited by 7 publications

(4 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The p1813-hPRL vector, considering that the characteristics of its elements allow high expression of recombinant proteins (Pereira et al 2014;Affonso et al 2018), had its efficiency evaluated in three strains, including RRI. The most efficient conditions were obtained with 0.1 mM IPTG and 9 h induction time (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Influence of The Expression Vector And of Its Elements On Recombinant hPRL Bacterial Synthesis: The Co-Directional Orientation of Replication And Transcription is Highly Critical

Affonso

Suzuki

Magalhães

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

The aim of the present work was to define a bacterial expression system that is particularly efficient for the synthesis of human prolactin (hPRL). In previous work, the synthesis of rec-hPRL by the p1813-hPRL vector in E. coli HB2151 was >500 mg/L, while it was much lower now (2.5-4-fold), in the strains RB791 and RRI. The highest positive influence on rec-hPRL synthesis was due to the transcription-replication co-orientation of hPRL cDNA and the ori/antibiotic resistance gene, responsible for up to a ~5-6-fold higher expression yield. In conclusion, this work confirmed that each bacterial strain of E. coli has a genetic set that can allow a different level of heterologous protein synthesis. The individual study of each element indicated that its action critically depends on the reading orientation in which it is located inside the vector: co-directional orientation of replication and transcription, in fact, greatly increased the level of rec-hPRL expression.

show abstract

Section: Resultsmentioning

confidence: 99%

Influence of The Expression Vector And of Its Elements On Recombinant hPRL Bacterial Synthesis: The Co-Directional Orientation of Replication And Transcription is Highly Critical

Affonso

Suzuki

Magalhães

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Circular dichroism spectrum (CD) is a useful tool to study protein refolding [17], thus we employed CD here to determine the secondary structure of the refolded ITGB1-head. The spectra showed a smooth curve with a negative peak at 205 nm (Fig.…”

Section: Expression Purification and Refolding Of Inclusion Bodiesmentioning

confidence: 99%

Expression, purification and renaturation of truncated human integrin β1 from inclusion bodies of Escherichia coli

Shi

Zhang

et al. 2015

Protein Expression and Purification

View full text Add to dashboard Cite

“…Ao final deste período a cultura foi diluída na proporção 1:20 em meio LB com canamicina e mantida nas mesmas condições de crescimento até a cultura bacteriana atingir uma densidade óptica (Do600~0,4-0,8) (Affonso, 2000). Nesta etapa se fez a ativação para a produção da proteína recombinante com a adição do reagente IPTG (isopropyl β-D-1-thiogalactopyranoside) em uma concentração final de 0,5M, por 5h a 37°C; 16h a 18°C, 25°C, 30°C e 37°C com agitação de 150 rpm (Pereira, 2014).…”

Section: Coluna Cromatográfiaunclassified

“…entre as sonicações, sempre em gelo. Essa suspensão foi centrifugada a 9.500 rpm por 10 min a 4°C, e coletado o sobrenadante contendo as proteínas solúveis (Pereira, 2014). Figura 12: Análise em gel de agarose 1% de clone positivo após reação de PCR utilizando primer T7 promotor e T7 terminador: (M) marcador de peso molecular Lambda EcoRI/HindIII, (1) amplificação a partir do vetor pET28_c-sECA.…”

Section: Extração Da Proteína Recombinanteunclassified

Obtenção, caractetizações estruturais e atividade enzimática do sítio C-catalítico da enzima conversora de angiotensina I - região ALA959 até SER1066

Elias¹

View full text Add to dashboard Cite

The angiotensin-converting enzyme (ACE) catalyzes the conversion of angiotensin I (Ang I) to the vasoconstrictor angiotensin II (Ang II) and the hydrolysis of bradykinin (BK). Human somatic ACE (sACE) has two homologous domains (N and C) that show 60% identity. Although these two regions have high homology, the catalytic site of the C-domain exhibits three times more activity than that of the N-domain in the hydrolysis of Ang I in vivo. This fact necessitates the development of new inhibitors or the improvement of existing ones. This study aimed to obtain the Ala 959 to Ser 1066 catalytic region of C-domain of sACE (c-sACE) in a structural conformation that resembles the native structure. We amplified the 324-bp sequence corresponding to the catalytic site of c-sACE and cloned this sequence into a pET28a(+) vector. The segment (named pET28_c-sACE) was expressed in a bacterial system. The expressed protein segment was soluble, and its purification was performed in one step using a His-tag affinity column. Structural analysis by circular dichroism and fluorescence confirmed that the purified protein is correctly folded, and an activity assay showed that c-sACE possesses enzymatic activity and is inhibited by lisinopril.

show abstract

A simple strategy for the purification of native recombinant full-length human RPL10 protein from inclusion bodies

Cited by 7 publications

References 31 publications

Influence of The Expression Vector And of Its Elements On Recombinant hPRL Bacterial Synthesis: The Co-Directional Orientation of Replication And Transcription is Highly Critical

Influence of The Expression Vector And of Its Elements On Recombinant hPRL Bacterial Synthesis: The Co-Directional Orientation of Replication And Transcription is Highly Critical

Expression, purification and renaturation of truncated human integrin β1 from inclusion bodies of Escherichia coli

Obtenção, caractetizações estruturais e atividade enzimática do sítio C-catalítico da enzima conversora de angiotensina I - região ALA959 até SER1066

Contact Info

Product

Resources

About