A New Strategy of Alternating Current Magnetic Field‐Assisted Three‐Phase Partitioning (ACMF‐TPP) for the Concentration and Partial Purification of Recombinant Prepromelittin Produced by <i>Komagataella phaffii</i>

The industrial-scale manufacturing and purification of melittin are of great interest due to its significant antibacterial and anticancer properties. Here, for the first time, prepromelittin (ppMEL), the precursor of melittin-a key component of bee venom-was extracellularly produced by Komagataella phaffii, and an alternating current magnetic field-assisted three-phase partitioning (ACMF-TPP) method was used to concentrate and partially purify the recombinant ppMEL (rppMEL). ACMF-TPP, a novel variation of the traditional TPP method widely used for biomolecular purification, was employed to purify recombinant protein from culture liquid more rapidly and in larger quantities. First, the amplified ppMEL gene was inserted into pPICZαA. So, the obtained pPICZαA-ppMEL was cloned into E. coli. Then, K. phaffii cells were transformed with linearized recombinant DNA, and a yeast clone was cultivated for extracellular rppMEL production. The optimal conditions of ACMF-TPP for the purification and concentration of rppMEL were the following parameters: pH 6.7, 70% w/v ammonium sulfate concentration, a 1:1 ratio of culture liquid to 2-propanol, 100% power, a 10-minute magnetic field duration, and a 70% duty cycle. As a result, it was observed that rppMEL primarily precipitated at the interface and that the difference in recovery efficiency (32.5% for TPP and 66.1% for ACMF-TPP, respectively) was statistically significant. The ACMF-TPP approach exhibited more efficacy than the TPP, demonstrating superior potential for separating and purifying other biomolecules. Moreover, this approach offers a cost-effective and scalable solution for the precise isolation, purification, and concentration of mixture components.