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Published online before print May 7, 2004, 10.1110/ps.03436604
Protein Science (2004), 13:1677-1683. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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FOR THE RECORD

Production of a fully functional, permuted single-chain penicillin G acylase

Gabriela Flores, Xavier Soberón and Joel Osuna

Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología/Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos 62250, México

(RECEIVED September 11, 2003; FINAL REVISION February 18, 2004; ACCEPTED February 26, 2004)



Abstract

Penicillin G acylase (PGA) is a heterodimeric enzyme synthesized as a single-polypeptide precursor that undergoes an autocatalytic processing to remove an internal spacer peptide to produce the active enzyme. We constructed a single-chain PGA not dependent on autoproteolytic processing. The mature sequence of the {beta}-domain was expressed as the N terminus of a new polypeptide, connected by a random tetra-peptide to the {alpha}-domain, to afford a permuted protein. We found several active enzymes among variants differing in their linker peptides. Protein expression analysis showed that the functional single-chain variants were produced when using a Sec-dependent leader peptide, or when expressed inside the bacterial cytoplasm. Active-site titration experiments showed that the single-chain proteins displayed similar kcat values to the ones obtained with the wild-type enzyme. Interestingly, the single-chain proteins also displayed close to 100% of functional active sites compared to 40% to 70% functional yield usually obtained with the heterodimeric protein.

Keywords: penicillin acylase; circular permutation; posttranslational processing; Tat secretion; Sec secretion

Reprint requests to: Joel Osuna, Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología/UNAM, Apdo. Postal 510-3, Cuernavaca, Morelos 62250, México; e-mail: joel{at}ibt.unam.mx; fax: (52) (777) 317 2388.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03436604.

Supplemental material: see www.proteinscience.org


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