ABSTRACT
BACKGROUND AND OBJECTIVES: Pfu DNA polymerase is an enzyme that exhibits the lowest error rate in the 3' to 5' exonuclease (proofreading) activity during DNA synthesis in Polymerase Chain Reactions (PCRs). This study was aimed to express and purify Pfu DNA polymerase in a bacterial expression system under a simple purification method. MATERIALS AND METHODS: Pfu polymerase gene sequence, derived from Pyrocuccus furiosus (Pfu) genomic DNA, was cloned and overexpressed in E. coli BL21 (DE3) pLysS. Upon overexpression, bacterial lysate containing the Pfu DNA polymerase was heated at 94°C for 5 minutes. Pfu DNA polymerase having high thermal stability was retained while the other bacterial proteins were denatured. The resulting thermo stable Pfu DNA polymerase was separated from the other debris of the denatured proteins by simple centrifugation. RESULTS: The enzymatic activity of the resulting Pfu DNA polymerase was estimated by comparing with the commercial Pfu DNA Polymerases. An estimated 50000 units of functional Pfu DNA polymerase was produced from a 400 ml culture. CONCLUSION: The in-house produced Pfu DNA Polymerase could be used for routine amplification that requires high-fidelity such as cloning and DNA sequencing.
ABSTRACT
Spheroids have been shown to recapitulate the tumour in vivo with properties such as the tumour microenvironment, concentration gradients, and tumour phenotype. As such, it can serve as a platform for determining the growth and invasion behaviour pattern of the cancer cells as well as be utilised for drug sensitivity assays; capable of exhibiting results that are closer to what is observed in vivo compared to two-dimensional (2D) cell culture assays. This study focused on establishing a three-dimensional (3D) cell culture model using the Nasopharyngeal Carcinoma (NPC) cell line, HK1 and analysing its growth and invasion phenotypes. The spheroids will also serve as a model to elucidate their sensitivity to the chemotherapeutic drug, Flavopiridol. The liquid overlay method was employed to generate the spheroids which was embedded in bovine collagen I matrix for growth and invasion phenotypes observation. The HK1 cells formed compact spheroids within 72 hours. Our observation from the 3 days experiments revealed that the spheroids gradually grew and invaded into the collagen matrix, showing that the HK1 spheroids are capable of growth and invasion. Progressing from these experiments, the HK1 spheroids were employed to perform a drug sensitivity assay using the chemotherapeutic drug, Flavopiridol. The drug had a dose-dependent inhibition on spheroid growth and invasion.
