Use of Rifampicin in T7 RNA Polymerase-Driven Expression of a Plant Enzyme: Rifampicin Improves Yield and Assembly

Kuderová, Alena; Nanak, Elizabeth; Truksa, Martin; Brzobohatý, Břetislav

doi:10.1006/prep.1999.1079

Cited by 16 publications

(8 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The addition of rifampicin at 15-20 min of postinduction with IPTG leads to inhibition of E. coli RNA polymerase, which in turn affects the production of the HCPs [39]. To each of the culture, rifampicin was added separately at the concentration of 50, 100, 150, 200, and 250 μg/ml after 15 min of IPTG (3 mM) induction.…”

Section: Effect Of Rifampicin On the Expression Of Rhifn-β-1bmentioning

confidence: 99%

Cloning, High Expression and Purification of Recombinant Human Intereferon-β-1b in Escherichia coli

Rao

Ramu

Rao

et al. 2008

Appl Biochem Biotechnol

View full text Add to dashboard Cite

Sequential evaluation and process control strategy were employed for impurity profile and high recovery with quality of rhIFN-beta-1b expressed in Escherichia coli. The high-level expression was achieved by using codon substitution (AT content of 52.6% at N-terminal region) and optimization of culture conditions. The addition of rifampicin at a concentration of 200 microg/ml has increased the specific product yield of 66 mg optical density(-1) l(-1) (43.5% of total cellular protein). Eighty-three percent of lipopolysaccharides, 32% of host deoxyribonucleic acid (DNA), and 78% of host cell proteins were removed by 0.75% Triton X-100 and 2 M urea wash. Eleven percent of lipopolysaccharides, 39% of host DNA, and 12% of host cell proteins were removed at the solubilization step. Ninety-two percent of protein refolding was achieved by high-pressure diafiltration method. Refolding by high-pressure diafiltration, bed height, and height equivalent to the theoretical plate value in chromatography column were identified as key parameters for high recovery with purity. Finally, the established process yielded 34% of purified protein with greater than 99% purity and is acceptable for preclinical toxicological studies. The purified rhIFN-beta-1b obtained in this study is the highest that has been reported so far.

show abstract

Section: Effect Of Rifampicin On the Expression Of Rhifn-β-1bmentioning

confidence: 99%

Cloning, High Expression and Purification of Recombinant Human Intereferon-β-1b in Escherichia coli

Rao

Ramu

Rao

et al. 2008

Appl Biochem Biotechnol

View full text Add to dashboard Cite

show abstract

“…We found that this test, although simple, is tedious and can fail to identify expressor colonies. However, there are a number of effective strategies that have been proposed over the years [2,3,6-8]. While we support these recommendations, in this work we sought to determine the underlying cause of reduced expression from pET vectors in order to more effectively prevent problems with poor protein production levels.…”

Section: Introductionmentioning

confidence: 99%

Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase

Vethanayagam

Flower

2005

Microb Cell Fact

View full text Add to dashboard Cite

Background: Protein expression vectors that utilize the bacteriophage T7 polymerase/promoter system are capable of very high levels of protein production. Frequently, however, expression from these vectors does not reliably achieve optimal levels of protein production. Strategies have been proposed previously that successfully maintain high expression levels, however we sought to determine the cause of induction failure.

show abstract

“…Moreover, in the T7 promoter/polymerase system, rifampicin can be used to selectively inhibit E. coli RNA polymerase-driven expression of endogenous host proteins (15). A dramatic reduction in endogenous protein synthesis that simplified isolation of (His) 6 Zm-p60.r expressed by the T7 RNA polymerase insensitive to rifampicin was observed (16).…”

Section: Discussionmentioning

confidence: 98%

Expression, Single-Step Purification, and Matrix-Assisted Refolding of a Maize Cytokinin Glucoside-Specific β-Glucosidase

Zouhar

Nanak

Brzobohatý

1999

Protein Expression and Purification

Self Cite

View full text Add to dashboard Cite

Use of Rifampicin in T7 RNA Polymerase-Driven Expression of a Plant Enzyme: Rifampicin Improves Yield and Assembly

Cited by 16 publications

References 30 publications

Cloning, High Expression and Purification of Recombinant Human Intereferon-β-1b in Escherichia coli

Cloning, High Expression and Purification of Recombinant Human Intereferon-β-1b in Escherichia coli

Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase

Expression, Single-Step Purification, and Matrix-Assisted Refolding of a Maize Cytokinin Glucoside-Specific β-Glucosidase

Contact Info

Product

Resources

About