Restoring 3′–5′ exonuclease activity of thermophilic Geobacillus DNA polymerase I using site-directed mutagenesis in active site

Rastgoo, Nasrin; Sadeghizadeh, Majid; Bambaei, B; Hosseinkhani, Saman

doi:10.1016/j.jbiotec.2009.09.006

Cited by 7 publications

(6 citation statements)

References 29 publications

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“…The pure enzyme Bst DNA polymerase has a specific activity of 813.56 U/mg. These results are in agreement with those reported by Rastgoo et al [7] that the specific activity of DNA polymerase from Geobacillus sp. MKK from wild-type and mutant DNA polymerase is in the range of 700 to more than 900 U/mg indicating that Bst DNA polymerase in this research has the potential to be developed.…”

Section: Introductionsupporting

confidence: 94%

Autoinduction expression of Bst DNA polymerase using lac operon-controlled expression systems in Escherichia coli

Laksmi,

Nuryana,

Vivid

et al. 2023

IOP Conf. Ser.: Earth Environ. Sci.

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Autoinduction simplified the steps for protein production by omitting the need to monitor the correct time for addition of inducer. This method is preferable for industrially production of protein. By using lac operon-controlled expression system, it is possible to produce the protein without the addition of IPTG as inducer. In a conventional way, IPTG was added at the exponential growth phase. By using autoinduction, IPTG could be replaced by lactose in the lac operon-controlled expression systems. Lactose was already supplemented in the medium and it was more cost efficient than IPTG. Therefore, this study aims to employ autoinduction method for expression of our protein target as it is a more convenient way and it is suitable for industrial application. In this study, DNA polymerase from Geobacillus stearothermophilus (Bst DNA polymerase), an enzyme which generally used in loop-mediated isothermal amplification (LAMP) assay, was expressed using lac operon-controlled expression systems in Escherichia coli. Bst DNA polymerase was successfully expressed using autoinduction expression method in LB medium containing 0.2% Lactose.

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Section: Introductionsupporting

confidence: 94%

Autoinduction expression of Bst DNA polymerase using lac operon-controlled expression systems in Escherichia coli

Laksmi,

Nuryana,

Vivid

et al. 2023

IOP Conf. Ser.: Earth Environ. Sci.

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“…The previous reports stated that the Bst DNA polymerase I Large Fragment has been cloned and 67.7kDa protein was expressed in E. coli cells for functional [20] and structural studies [21]. On the other hand, recently in year 2009 the full length gene of Bst DNA polymerase was successfully cloned into pET28a(+) vector and the recombinant proteins were produced [22]. The recombinant proteins with similar molecular weight as stated by Rastgoo were successful produced in this current study.…”

Section: Discussionsupporting

confidence: 56%

“…They have been reported that gene encoding for DNA polymerase in thermophilic Geobacillus sp. MKK successfully expressed with 0.1 mM IPTG [22]. Initially, the similar condition was used for expression in this study.…”

Section: Discussionmentioning

confidence: 99%

PCR-based Gene Synthesis, Cloning, Expression, Purification and Characterization of Bst DNA Polymerase in E. coli Cells

Suppan¹,

Shamsuddin²,

Ismail³

et al. 2015

Curr Synthetic Sys Biol

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Gene synthesis is a technique for modifying genes for studying gene function, structure and expression. There are several PCR based strategies and non-PCR based strategies for gene synthesis. Here we perform the 2 step gene synthesis combining assembly PCR and sequential overlap extension PCR strategies to synthesize the full length of Bst DNA polymerase (2,648bp) and express the protein in E. coli cells. The full length Bst DNA polymerase was divided into 5 short DNA Fragments and the oligonucloetides were designed for the entire sequence each with 40-45 mers. In step 1, the oligonuleotides of each fragment were assembled and then the gene fragment was amplified separately through assembly PCR method. In step 2, sequentially the two adjacent fragments were assembled through overlap extension PCR at a time until the full length gene completely synthesized and finally cloned into pCR®2.1-TOPO vector. Then the full length Bst DNA pol gene was subcloned into pET28a(+) expression vector and finally expressed in BL21(DE3) E. coli cells. The purified protein was identified by MALDITOF analysis. The polymerization activity of the recombinant Bst DNA polymerase was compared with commercial enzyme. The 98kDA recombinant Bst DNA polymerase succeed in amplification of 100bp DNA fragment via helicase dependent amplification (HDA).

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“…In recent years, researchers have tried to address this problem by selecting an exonuclease‐deficient host strain or inactivating the nuclease in the host strain by random mutations to prevent the digestion of mRNAs by 3′–5′ exonucleases (Rastgoo, Sadeghizadeh, Bambaei, & Hosseinkhani, ; Zhang, McCorkle, Novak, Yang, & Kaetzel, ). However, both of these two methods could affect the normal physiological activity of cells.…”

Section: Introductionmentioning

confidence: 99%

Synthetic repetitive extragenic palindromic (REP) sequence as an efficient mRNA stabilizer for protein production and metabolic engineering in prokaryotic cells

Deng

Liu

et al. 2018

Biotech & Bioengineering

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In prokaryotic cells, 3'-5' exonucleases can attenuate messenger RNA (mRNA) directionally from the direction of the 3'-5' untranslated region (UTR), and thus improving the stability of mRNAs without influencing normal cell growth and metabolism is a key challenge for protein production and metabolic engineering. Herein, we significantly improved mRNA stability by using synthetic repetitive extragenic palindromic (REP) sequences as an effective mRNA stabilizer in two typical prokaryotic microbes, namely, Escherichia coli for the production of cyclodextrin glucosyltransferase (CGTase) and Corynebacterium glutamicum for the production of N-acetylglucosamine (GlcNAc). First, we performed a high-throughput screen to select 4 out of 380 REP sequences generated by randomizing 6 nonconservative bases in the REP sequence designed as the degenerate base "N." Secondly, the REP sequence was inserted at several different positions after the stop codon of the CGTase-encoding gene. We found that mRNA stability was improved only when the space between the REP sequence and stop codon was longer than 12 base pairs (bp). Then, by reconstructing the spacer sequence and secondary structure of the REP sequence, a REP sequence with 8 bp in a stem-loop was obtained, and the CGTase activity increased from 210.6 to 291.5 U/ml. Furthermore, when this REP sequence was added to the 3'-UTR of glucosamine-6-phosphate N-acetyltransferase 1 ( GNA1), which is a gene encoding a key enzyme GNA1 in the GlcNAc synthesis pathway, the GNA1 activity was increased from 524.8 to 890.7 U/mg, and the GlcNAc titer was increased from 4.1 to 6.0 g/L in C. glutamicum. These findings suggest that the REP sequence plays an important function as an mRNA stabilizer in prokaryotic cells to stabilize its 3'-terminus of the mRNA by blocking the processing action of the 3'-5' exonuclease. Overall, this study provides new insight for the high-efficiency overexpression of target genes and pathway fine-tuning in bacteria.

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Restoring 3′–5′ exonuclease activity of thermophilic Geobacillus DNA polymerase I using site-directed mutagenesis in active site

Cited by 7 publications

References 29 publications

Autoinduction expression of Bst DNA polymerase using lac operon-controlled expression systems in Escherichia coli

Autoinduction expression of Bst DNA polymerase using lac operon-controlled expression systems in Escherichia coli

PCR-based Gene Synthesis, Cloning, Expression, Purification and Characterization of Bst DNA Polymerase in E. coli Cells

Synthetic repetitive extragenic palindromic (REP) sequence as an efficient mRNA stabilizer for protein production and metabolic engineering in prokaryotic cells

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