RESUMEN
Autoinduction systems in Escherichia coli can control the production of proteins without the addition of a particular inducer. In the present study, we optimized the heterologous expression of Moloney Murine Leukemia Virus derived Reverse Transcriptase (MMLV-RT) in E. coli. Among 4 autoinduction media, media Imperial College resulted the highest MMLV-RT overexpression in E. coli BL21 Star (DE3) with incubation time 96 h. The enzyme was produced most optimum in soluble fraction of lysate cells. The MMLV-RT was then purified using the Immobilized Metal Affinity Chromatography method and had specific activity of 629.4 U/mg. The system resulted lower specific activity and longer incubation of the enzyme than a classical Isopropyl ß-D-1-thiogalactopyranoside (IPTG)-induction system. However, the autoinduction resulted higher yield of the enzyme than the conventional induction (27.8%). Techno Economic Analysis revealed that this method could produce MMLV-RT using autoinduction at half the cost of MMLV-RT production by IPTG-induction. Bioprocessing techniques are necessary to conduct to obtain higher quality of MMLV-RT under autoinduction system.
Asunto(s)
Escherichia coli , Virus de la Leucemia Murina de Moloney , ADN Polimerasa Dirigida por ARN , Escherichia coli/genética , Escherichia coli/metabolismo , Virus de la Leucemia Murina de Moloney/genética , Virus de la Leucemia Murina de Moloney/enzimología , ADN Polimerasa Dirigida por ARN/metabolismo , ADN Polimerasa Dirigida por ARN/genética , Isopropil Tiogalactósido/farmacología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Medios de CultivoRESUMEN
A DNA polymerase from Thermus aquaticus remains the most popular among DNA polymerases. It was widely applied in various fields involving the application of polymerase chain reaction (PCR), implying the high commercial value of this enzyme. For this reason, an attempt to obtain a high yield of Taq DNA polymerase is continuously conducted. In this study, the l-rhamnose-inducible promoter rhaBAD was utilized due to its ability to produce recombinant protein under tight control in E. coli expression system. Instead of full-length Taq polymerase, an N-terminal deletion of Taq polymerase was selected. To obtain a high-level expression, we attempted to optimize the codon by reducing the rare codon and GC content, and in a second attempt, we optimized the culture conditions for protein expression. The production of Taq polymerase using the optimum culture condition improved the level of expression by up to 3-fold. This approach further proved that a high level of recombinant protein expression could be achieved by yielding a purified Taq polymerase of about 8.5 mg/L of culture. This is the first research publication on the production of Taq polymerase with N-terminal deletion in E. coli with the control of the rhaBAD promoter system.
Asunto(s)
Codón , Escherichia coli , Regiones Promotoras Genéticas , Proteínas Recombinantes , Polimerasa Taq , Escherichia coli/genética , Escherichia coli/metabolismo , Codón/genética , Polimerasa Taq/metabolismo , Polimerasa Taq/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Thermus/genética , Thermus/enzimología , Secuencia de BasesRESUMEN
BACKGROUND: DNA polymerase is an essential component in PCR assay for DNA synthesis. Improving DNA polymerase with characteristics indispensable for a powerful assay is crucial because it can be used in wide-range applications. Derived from Pyrococcus furiosus, Pfu DNA polymerase (Pfu pol) is one of the excellent polymerases due to its high fidelity. Therefore, we aimed to develop Pfu pol from a synthetic gene with codon optimization to increase its protein yield in Escherichia coli. RESULTS: Recombinant Pfu pol was successfully expressed and purified with a two-step purification process using nickel affinity chromatography, followed by anion exchange chromatography. Subsequently, the purified Pfu pol was confirmed by Western blot analysis, resulting in a molecular weight of approximately 90 kDa. In the final purification process, we successfully obtained a large amount of purified enzyme (26.8 mg/L). Furthermore, the purified Pfu pol showed its functionality and efficiency when tested for DNA amplification using the standard PCR. CONCLUSIONS: Overall, a high-level expression of recombinant Pfu pol was achieved by employing our approach in the present study. In the future, our findings will be useful for studies on synthesizing recombinant DNA polymerase in E. coli expression system.
RESUMEN
Bst DNA polymerase is a DNA polymerase derived from Geobacillus stearothermophilus, has a strand-displacement activity, and is used in loop-mediated isothermal amplification (LAMP) for rapid detection of COVID-19. Despite its potential to be employed in the detection of COVID-19, using commercially available enzymes is not economically feasible. The use of noncommercial enzyme for routine use is desirable. However, research on Bst DNA polymerase is still limited in Indonesia. For those reasons, a preliminary study of scale-up production of recombinant Bst polymerase was conducted. Therefore, the optimization of expression conditions was performed. The optimum conditions for Bst polymerase expression were as follows: 1 mM of IPTG, post-induction incubation time of 6 h, and induction at OD600 1.1. Employing optimum conditions could result in 2.8 times increase in protein yield compared to the initial conditions. Subsequently, an operation in 1 L working volume by a lab-scale bioreactor had been performed, followed by purification and dialysis. The optimum result for a 1 L lab-scale bioreactor was achieved by applying 100 rpm and 3 vvm, giving 11.7 mg/L of protein yield. Bst polymerase was successfully purified showing 813.56 U/mg of polymerase activity.
Asunto(s)
COVID-19 , ADN Polimerasa I , Humanos , Geobacillus stearothermophilus/genética , Replicación del ADN , Escherichia coli/genéticaRESUMEN
Cervical cancer caused by Human papillomavirus (HPV) is one of the most common causes of cancer death in women worldwide. Even though the disease can be avoided by immunization, the expensive price of HPV vaccines makes it hard to be accessed by women in middle-low-income countries. Thus, the development of generic HPV vaccines is needed to address inequalities in life-saving access. This study aimed to develop the HPV52 L1 VLP-based recombinant vaccine using Pichia pastoris expression system. The l1 gene was codon-optimized based on P. pastoris codon usage resulting CAI value of 0.804. The gene was inserted into the pD902 plasmid under the regulation of the AOX1 promoter. The linear plasmid was transformed into P. pastoris BG10 genome and screened in YPD medium containing zeocin antibiotic. Colony of transformant that grown on highest zeocin concentration was characterized by genomic PCR and sequencing. The positive clone was selected and expressed using BMGY/BMMY medium induced with various methanol concentrations. The SDS-PAGE and Western blot analyses showed that 55 kDa L1 protein was successfully expressed using an optimum concentration of 1% methanol. The self-assembly of HPV52 L1 protein was also proven using TEM analysis. Moreover, we also analyzed the B-cell epitope of HPV52 L1 protein based on several criteria, including antigenicity, surface accessibility, flexibility, and hydrophilicity. We assumed that epitope 476GLQARPKLKRPASSAPRTSTKKKKV500 could be developed as an epitope-based vaccine with a neutralizing antibody response toward HPV52 infection. Finally, our study provided the alternative for developing low-cost HPV vaccines, either VLP or epitope-based.
Asunto(s)
Virus del Papiloma Humano , Vacunas contra Papillomavirus , Femenino , Humanos , Metanol/metabolismo , Proteínas de la Cápside/genética , Pichia/genética , Pichia/metabolismo , Vacunas contra Papillomavirus/genética , Vacunas contra Papillomavirus/metabolismo , Epítopos/metabolismo , Codón/metabolismoRESUMEN
Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is the most frequently used enzyme in molecular biology for cDNA synthesis. To date, reverse transcription coupled with Polymerase Chain Reaction, known as RT-PCR, has been popular as an excellent approach for the detection of SARS-CoV-2 during the COVID-19 pandemic. In this study, we aimed to improve the enzymatic production and performance of MMLV-RT by optimizing both codon and culture conditions in E. coli expression system. By applying the optimized codon and culture conditions, the enzyme was successfully overexpressed and increased at high level based on the result of SDS-PAGE and Western blotting. The total amount of MMLV-RT has improved 85-fold from 0.002 g L-1 to 0.175 g L-1 of culture. One-step purification by nickel affinity chromatography has been performed to generate the purified enzyme for further analysis of qualitative and quantitative RT activity. Overall, our investigation provides useful strategies to enhance the recombinant enzyme of MMLV-RT in both production and performance. More importantly, the enzyme has shown promising activity to be used for RT-PCR assay.
Asunto(s)
Virus de la Leucemia Murina de Moloney , Codón/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Virus de la Leucemia Murina de Moloney/enzimología , Virus de la Leucemia Murina de Moloney/genética , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismoRESUMEN
Purpose: Strategy for improving the production of biopharmaceutical protein continues to develop due to increasing market demand. Human granulocyte colony stimulating factor (hG-CSF) is one of biopharmaceutical proteins that has many applications, and easily produced in Escherichia coli expression system. Previous studies reported that codon usage, rare codon, mRNA folding and GC-content at 5'-terminal end were crucial for protein production in E. coli. In the present study, the effect of reducing the GC-content and increasing the mRNA folding free energy at the 5'-terminal end on the expression level of hG-CSF proteins was investigated. Methods: Synonymous codon substitutions were performed to generate mutant variants of open reading frame (ORF) with lower GC-content at 5'-terminal ends. Oligoanalyzer tool was used to calculate the GC content of eight codons sequence after ATG. Whereas, mRNA folding free energy was predicted using KineFold and RNAfold tools. The template DNA was amplified using three variant forward primers and one same reverse primer. Those DNA fragments were individually cloned into pJexpress414 expression vector and were confirmed using restriction and DNA sequencing analyses. The confirmed constructs were transformed into E. coli NiCo21(DE3) host cells and the recombinant protein was expressed using IPTG-induction. Total protein obtained were characterized using SDS-PAGE, Western blot and ImageJ software analyses. Results: The result showed that the mutant variant with lower GC-content and higher mRNA folding free energy near the translation initiation region (TIR) could produce a higher amount of hG-CSF proteins compared to the original gene sequence. Conclusion: This study emphasized the important role of the nucleotide composition immediately downstream the start codon to achieve high-yield protein product on heterologous expression in E. coli.
RESUMEN
In the previous study, we constructed an expression vector carrying the anti-EGFRvIII scFv antibody gene with VH-linker-VL orientation. The proteins were successfully produced in the periplasmic space of Escherichia coli. In this study, we substituted the inserted DNA with VL-linker-VH orientation of the anti-EGFRvIII scFv gene and analyzed its expression in E. coli. The DNA fragment was amplified from its cloning vector (pTz-rscFv), subsequently cloned into a previous expression vector containing the pelB signal sequence and his-tag, and then transformed into E. coli TOP10. The recombinant plasmids were characterized by restriction, PCR, and DNA sequencing analyses. The new anti-EGFRvIII scFv antibody proteins have been successfully expressed in the periplasmic compartment of E. coli Nico21(DE3) using 0.1 mM final concentration of IPTG induction. Total proteins, soluble periplasmic and cytoplasmic proteins, solubilized inclusion bodies, and extracellular proteins were analyzed by SDS-PAGE and Western Blot analyses. The results showed that soluble scFv proteins were found in all fractions except from the cytoplasmic space.