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Here, for the first time, prepromelittin (ppMEL), the precursor of melittin\u2014a key component of bee venom\u2014was extracellularly produced by\n                    <jats:italic>Komagataella phaffii<\/jats:italic>\n                    , and an alternating current magnetic field\u2010assisted three\u2010phase partitioning (ACMF\u2010TPP) method was used to concentrate and partially purify the recombinant ppMEL (rppMEL). ACMF\u2010TPP, 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\u00a0amplified ppMEL gene was inserted into pPICZ\u03b1A. So, the obtained pPICZ\u03b1A\u2010ppMEL was cloned into\n                    <jats:italic>E. coli<\/jats:italic>\n                    . Then,\n                    <jats:italic>K. phaffii<\/jats:italic>\n                    cells were transformed with linearized recombinant DNA, and a yeast clone was cultivated for extracellular rppMEL production. The optimal conditions of ACMF\u2010TPP 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\u2010propanol, 100% power, a 10\u2010minute 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\u2010TPP, respectively) was statistically significant. The ACMF\u2010TPP approach exhibited more efficacy than the TPP, demonstrating superior potential for separating and purifying other biomolecules. Moreover, this approach offers a cost\u2010effective and scalable solution for the precise isolation, purification, and concentration of mixture components.\n                  <\/jats:p>","DOI":"10.1002\/bab.70126","type":"journal-article","created":{"date-parts":[[2026,1,7]],"date-time":"2026-01-07T06:16:03Z","timestamp":1767766563000},"page":"1728-1738","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["A New Strategy of Alternating Current Magnetic Field\u2010Assisted Three\u2010Phase Partitioning (ACMF\u2010TPP) for the Concentration and Partial Purification of Recombinant Prepromelittin Produced by\n                    <i>Komagataella phaffii<\/i>"],"prefix":"10.1002","volume":"73","author":[{"given":"Seyda Yildiz","family":"Arslan","sequence":"first","affiliation":[{"name":"Department of Molecular Biology and Genetics, Institute of Science and Technology Atat\u00fcrk University  Erzurum 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