PHUSION DNA POLYMERASE PRODUCTION IN Escherichia coli USING LACTOSE AS INDUCER IS A LOW COST ALTERNATIVE TO IPTG

Polymerase chain reaction (PCR) is an essential technique in molecular biology and relies on thermostable DNA polymerases, including high-fidelity enzymes such as Phusion DNA polymerase. However, the cost of recombinant enzyme production can limit its routine use, particularly in research and teaching laboratories. In this study, we evaluated lactose as a low-cost alternative to IPTG for the induction of recombinant Phusion DNA polymerase production in Escherichia coli. Bacterial cultures were grown in LB medium and induced at an optical density (OD₆₀₀) between 0.5 and 0.8 using either 0.5 mM IPTG or 1% lactose. Expression experiments were performed in shake flasks and in a 5-L bioreactor (Eppendorf BioFlo 320), with bacterial growth and protein production monitored over time. Recombinant Phusion polymerase was subsequently purified and characterized by protein quantification, SDS-PAGE, and functional PCR assays. No significant difference in protein production was observed between lactose and IPTG induction in shake-flask cultures. In contrast, bioreactor cultivation resulted in significantly higher protein concentrations with lactose induction (27.41 mg/mL) than with IPTG (16.36 mg/mL), corresponding to an approximately 68% increase in protein yield. Furthermore, the estimated inducer cost was approximately US$0.74/L of culture for 1% lactose compared with US$7.36/L for 0.5 mM IPTG, representing an approximately 90% reduction in inducer cost per liter of culture. When the inducer cost was normalized to the relative protein production obtained in the bioreactor, lactose resulted in an estimated 94% reduction in inducer-associated cost per unit of recombinant protein produced compared with IPTG. SDS-PAGE analysis revealed a band at approximately 97 kDa, consistent with the expected molecular mass of Phusion polymerase, and the purified enzyme successfully amplified a 1,756-bp DNA fragment, confirming its functionality. Further characterization is being performed by comparison with commercial Phusion DNA polymerase. Overall, these results demonstrate that lactose can simultaneously increase recombinant Phusion polymerase production and substantially reduce induction costs, supporting its use as an economically attractive alternative to IPTG for enzyme production.