An Application of pET SUMO Protein Expression System in Escherichia coli: Cloning, Expression, Purification, and Characterisation of Native Kras4BG12V Oncoprotein

Tan, Michelle Siying; Teh, Yuan Han; Ho, Ken; Stanslas, Johnson

doi:10.1007/s10930-019-09872-1

Cited by 7 publications

(4 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This study showed that the recombinant protein with fusion tag remained in the resin when elution was done by 250 mM imidazole. It was hypothesized that poly histidine tag promotes oligomerization of the recombinant protein, which indicates higher imidazole concentration is needed to elute the protein [ 7 ]. Since the washing process is composed a low imidazole, the steps should be optimized to maintain the target protein.…”

Section: Discussionmentioning

confidence: 99%

“…Then, 1% of pre-cultured bacteria was inoculated into a fresh LB medium and incubated at 37 °C for ± 3 h, until OD 600 reached ~0.5–0.6, which is a logarithmic phase of the E. coli BL21 DE3. The culture induction used 0.5 mM IPTG along with an addition of 1% (v/v) glucose and incubated at 20 °C for ± 5 h, until the OD 600 reached ~1.0 as the stationary phase of the E. coli BL21 DE3 [ 7 , 9 ]. The bacterial cells were harvested by centrifugation at 6000 rpm for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…Formation of inclusion bodies in bacterial hosts poses a major challenge for large-scale production [ 6 ]. The SUMO fusion protein is a small ubiquitin-related modifier that enhances the solubility of the expressed recombinant protein [ 7 ]. This study used a codon-optimized L1 HPV 52 to induce higher expression in E. coli BL21 DE3 that is composed of a Champion pET SUMO protein expression system.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Construction, expression, and in vitro assembly of virus-like particles of L1 protein of human papillomavirus type 52 in Escherichia coli BL21 DE3

Mustopa

Meilina

Irawan

et al. 2022

Journal of Genetic Engineering and Biotechnology

View full text Add to dashboard Cite

Background A major discovery in human etiology recognized that cervical cancer is a consequence of an infection caused by some mucosatropic types of human papillomavirus (HPV). Since L1 protein of HPV is able to induce the formation of neutralizing antibodies, it becomes a protein target to develop HPV vaccines. Therefore, this study aims to obtain and analyze the expression of HPV subunit recombinant protein, namely L1 HPV 52 in E. coli BL21 DE3. The raw material used was L1 HPV 52 protein, while the synthetic gene, which is measured at 1473 bp in pD451-MR plasmid, was codon-optimized (ATUM) and successfully integrated into 5643 base pairs (bps) of pETSUMO. Bioinformatic studies were also conducted to analyze B cell epitope, T cell epitope, and immunogenicity prediction for L1HPV52 protein. Results The pETSUMO-L1HPV52 construct was successfully obtained in a correct ligation size when it was cut with EcoRI. Digestion by EcoRI revealed a size of 5953 and 1160 bps for both TA cloning petSUMO vector and gene of interest, respectively. Furthermore, the right direction of construct pETSUMO-L1HPV52 was proven by PCR techniques using specific primer pairs then followed by sequencing, which shows 147 base pairs. Characterization of L1 HPV 52 by SDS-PAGE analysis confirms the presence of a protein band at a size of ~55 kDa with 6.12 mg/L of total protein concentration. Observation under by transmission electron microscope demonstrates the formation of VLP-L1 at a size between 30 and 40 nm in assembly buffer under the condition of pH 5.4. Based on bioinformatics studies, we found that there are three B cell epitopes (GFPDTSFYNPET, DYLQMASEPY, KEKFSADLDQFP) and four T cell epitopes (YLQMASEPY, PYGDSLFFF, DSLFFFLRR, MFVRHFFNR). Moreover, an immunogenicity study shows that among all the T cell epitopes, the one that has the highest affinity value is DSLFFFLRR for Indonesian HLAs. Conclusion Regarding the achievement on successful formation of L1 HPV52-VLPs, followed by some possibilities found from bioinformatics studies, this study suggests promising results for future development of L1 HPV type 52 vaccine in Indonesia.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Construction, expression, and in vitro assembly of virus-like particles of L1 protein of human papillomavirus type 52 in Escherichia coli BL21 DE3

Mustopa

Meilina

Irawan

et al. 2022

Journal of Genetic Engineering and Biotechnology

View full text Add to dashboard Cite

show abstract

“…The addition of fusion tags is one of the various strategies for enhancing recombinant protein solubility and protein expression efficiency [40]. In the previous studies, fusion tags, namely, glutathione S-transferase (GST), were applied in the production of recombinant GRA7 protein as antigen for serodiagnosis of T. gondii infection in cats [26,32] while a small ubiquitin-related modifier (SUMO) [39,41,42] has never been used to produce recombinant GRA7 protein in the detection of T. gondii infection in cats.…”

Section: Introductionmentioning

confidence: 99%

Development and evaluation of indirect enzyme-linked immunosorbent assay using recombinant dense granule antigen 7 protein for the detection of Toxoplasma gondii infection in cats in Thailand

et al. 2022

View full text Add to dashboard Cite

Background and Aim: Toxoplasma gondii is recognized as a zoonosis causing toxoplasmosis in animals globally. Cat is a definitive host of T. gondii and sheds oocyst through feces, which can infect human beings and animals through contaminated food ingestion. A precise diagnostic test is essential to prevent T. gondii infection in both humans and animals. This study aimed to develop and evaluate the pETite- dense granule antigen 7(GRA7)-based indirect enzyme-linked immunosorbent assay (ELISA) to detect T. gondii infection in cats. Materials and Methods: T. gondii-GRA7 was cloned and expressed in the Expresso® small ubiquitin-related modifier (SUMO) T7 Cloning and Expression System. The recombinant pETite-GRA7 was purified using HisTrap affinity chromatography and confirmed using Western blot analysis. The recombinant protein was used to develop and evaluate the indirect ELISA for T. gondii infection detection. In total, 200 cat sera were tested using pETite-GRA7-based indirect ELISA and indirect fluorescent antibody test (IFAT). The statistical analysis based on Kappa value, sensitivity, specificity, positive predictive value, negative predictive value, χ2 test, and receiver operating characteristic (ROC) curve was used to evaluate the performance of the test. Results: A 606 bp GRA7 polymerase chain reaction (PCR) product was obtained from T. gondii RH strain genomic DNA. The gene was cloned into the pETite™ vector and transformed to HI-Control Escherichia coli BL21 (DE3) for protein expression. Approximately 35 kDa of recombinant pETite-GRA7 was observed and Western blot analysis showed positive bands against anti-6-His antibody and positive-T. gondii cat serum. A sample of 0.5 μg/mL of pETite-GRA7 was subjected to indirect ELISA to detect T. gondii infection in the cat sera. The results showed sensitivity and specificity of pETite- GRA7-based indirect ELISA at 72% and 96%, respectively. An acceptable diagnostic performance was characterized by high concordant results (94%) and substantial agreement (Kappa value=0.65) with IFAT. The seroprevalence levels of ELISA and IFAT were 10% and 9%, respectively, and were not significantly (p>0.05) different. The expected performance of ELISA at different cutoff points using the ROC curve analysis revealed 89% sensitivity and 92% specificity at the cutoff value of 0.146, with a high overall assay accuracy (area under the curve=0.94). Conclusion: In this study, the pETite™ vector, N-terminal 6xHis SUMO fusion tag, was used to improve the solubility and expression level of GRA7. The recombinant pETite-GRA7 showed enhanced protein solubility and purification without special condition requirements. This pETite-GRA7-based indirect ELISA showed high concordant results and substantial agreement with IFAT. ELISA revealed an acceptable sensitivity and specificity. These initial data obtained from cats' sera demonstrated that pETite-GRA7-based indirect ELISA could be a useful method for local serological diagnosis of T. gondii infection in cats in Thailand.

show abstract

Tagging Recombinant Proteins to Enhance Solubility and Aid Purification

Loughran,

Walls

2023

Methods in Molecular Biology

View full text Add to dashboard Cite

An Application of pET SUMO Protein Expression System in Escherichia coli: Cloning, Expression, Purification, and Characterisation of Native Kras4BG12V Oncoprotein

Cited by 7 publications

References 15 publications

Construction, expression, and in vitro assembly of virus-like particles of L1 protein of human papillomavirus type 52 in Escherichia coli BL21 DE3

Construction, expression, and in vitro assembly of virus-like particles of L1 protein of human papillomavirus type 52 in Escherichia coli BL21 DE3

Development and evaluation of indirect enzyme-linked immunosorbent assay using recombinant dense granule antigen 7 protein for the detection of Toxoplasma gondii infection in cats in Thailand

Tagging Recombinant Proteins to Enhance Solubility and Aid Purification

Contact Info

Product

Resources

About