Optimization of Osteopontin Recombinant Protein as a Candidate Supplementation for Semen Preservation

Abstract

The recombinant protein of heterologous proteins in Escherichia coli strains K12 has various and different systems tested and demands a detailed insight into the multiple factors affecting the encoded protein. One of the crucial factors is the acceptable quality of the DNA copies inserted inside the bacteria. Firstly, the amplification procedure needed to be performed well; thus, designing the primer and selecting the optimum annealing temperature are the focus indicators in this study. This study obtained a reference gene from the NCBI data bank with Reference Sequence: NM_174187.2. Two types of primers (SPP1FSPP1R and OPN1F - OPN1R) with different targeted bands were designed and selected after being reconstructed using the software. Online software such as addgene.org is also used to identify the right restriction site. The annealing temperature distinguished the PCR system used to amplify each primer. The result of this study revealed the best annealing at 65ºC successfully amplified 820 bp of the targeted band. The phenomenon not following the theory of blue-white screening is the empty plasmid control, where not a single colony grows on the media. Competent cells inserted with empty plasmids should still be able to expand on LB-Amp agar media because the presence of these plasmids is capable of providing resistance to antibiotics (in this case, ampicillin). This discrepancy is thought to have been caused by the improper insertion of the empty plasmid so that the plasmid did not enter the competent cell. Key words: Osteopontin, Primer, DNA clone, PCR amplification