This study aimed to optimize the expression of the CD80 protein in Escherichia coli BL21 (DE3) using the high copy pET32C+ plasmid system. CD80, a key costimulatory molecule in immune response, poses significant challenges in recombinant expression, primarily due to issues related to protein solubility and plasmid stability. This research focused on refining colony incubation conditions for colony PCR to address plasmid stability concerns, as well as optimizing induction parameters to enhance both protein yield and solubility. Initial transformations of the ligated CD80 sequence into DH5α cells were successful, but subsequent transformations into BL21 cells necessitated further optimization due to nonspecific binding observed in colony PCR and reduced plasmid stability. The findings revealed that post-transformation incubation at 30 °C significantly improved plasmid stability compared to 37 °C, thereby minimizing nonspecific binding. Furthermore, induction at 18 °C was found to produce soluble CD80 protein at lower IPTG concentrations, in contrast to the higher IPTG requirements at 28 °C to achieve similar solubility. Despite these optimizations, the expressed CD80 protein was truncated, likely due to the presence of rare codons in BL21. It is recommended that future studies utilize the BL21 CodonPlus strain to facilitate the expression of full-length CD80, providing valuable insights for improving recombinant protein production in E. coli.