Optimized expression of large fragment DNA polymerase I from <i>Geobacillus stearothermophilus</i> in <i>Escherichia coli</i> expression system

Nuryana, Isa; Laksmi, Fina Amreta; Dewi, Kartika Sari; Wijaya, Hans; Rahmani, Nanik; Yudiargo, Danu Risqi; Ismadara, Astadewi; Helbert,; Hadi, Moch. Irfan; Purnawan, Awan; Djohan, Apridah Cameliawati

doi:10.1080/10826068.2022.2095573

Cited by 8 publications

(6 citation statements)

References 25 publications

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“…Among other complex media, LB was selected as the growth medium in this present study as it was reported as the common complex medium for expressing proteins in E. coli by autoinduction method by up to 60% [19]. In this present study, large fragment Bst DNA polymerase that was constructed in the previous study was utilized [6]. For the purpose of in-house production of Bst DNA polymerase in Indonesia, the enzyme was produced in the T7 lac promoter-based expression system in E. coli by autoinduction method.…”

Section: Resultsmentioning

confidence: 99%

“…Highlevel expression of various proteins using autoinduction method were previously reported [12][13][14]. In this study, large fragment Bst DNA polymerase from previous report by Agustriana et al [6] was used and it was successfully expressed using autoinduction method. The yield of the enzyme and the bacterial biomass were investigated when several complex media were used and thereby can provide insight in the production of Bst DNA polymerase by utilizing autoinduction method in complex media.…”

Section: Introductionmentioning

confidence: 93%

“…For protein expression, E. coli BL21 Star TM (DE3) were utilized. The expression vector, pD451-SR (ATUM) containing T7 promoter, was utilized to create the plasmid pD451-SR_Bstpol, which included Bst DNA polymerase [6]. Colonies carrying an expression vector were pre-cultured in Luria-Bertani medium (LB) with 0.4% glucose and 30 mg/L kanamycin.…”

Section: Strain Vector and Mediummentioning

confidence: 99%

“…By examining the target protein's band and comparing it to the linear equation generated from the BSA standard curve, the yield of Bst DNA polymerase was calculated. The ImageJ analysis program was used to complete this task [6].…”

Section: Determination Of Bst Dna Polymerase Yieldmentioning

confidence: 99%

“…In previous investigation by Agustriana et al [6], the construction of the gene encoding Bst DNA polymerase has been carried out. The gene was successfully cloned into the plasmid pD451-SR and transformed into E. coli BL21 star (DE3).…”

Section: Introductionmentioning

confidence: 99%

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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: Resultsmentioning

confidence: 99%