Molecular Microbiology (1988) 2(4), 519-525 Overexpression, solubiiization and refolding of a genetically engineered derivative of the penicillin- binding protein 3 of Escherichia coli K12 J. Bartholome-De Belder,^ M. Nguyen-Disteche,^ N. Houba-Herin,^* J. M. Ghuysen,^^ I. N. Maruyatna,^^ H. Hara,^ Y. Hirota,^ and M. Inouye^* ^Service de Microbiologie, Universite de Liege, Instit de Chimie, B6. B-4000 Sart Tilman (Liege 1), Belgium. ^Microbial Genetics. National Institute of Genetics, Yata-1, 111 fvlishima, Shizuoka-ken, Japan. ^Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, New Jersey 08854-5635, USA. Summary Replacement of the amino-terminal 40-amino-acid region of the 588-ammo-acid precursor of the mem- brane-bound penicillin-binding protein 3 (P6P3) by the decapeptide MKGKEFQAWI was carried out by altering the amino-coding end of the ftsi gene. Inser- tion of the modified gene into a runaway-replication plasmid under the control of a fused Ipp promoter and lac promoter/operator, resulted in the overexpression by Escherichia coli of the modified PBP3 (designated PBP3**) in the cytoplasm. About 80% of the accumu- lated PBP3** underwent sequestration in the form of insoluble protein granules that were isolated by cell breakage or cell lysis. After selective removal of contaminants by an EDTA-lysozyme/DNase (de- oxyribonuclease)/Nonidet extraction, treatment of the granules with guanidinium chloride followed by dialy- sis against buffer containing 0.5 M NaCI yielded a refolded, water-soluble PBP3**, which, upon chroma- tography on Superose 12, exhibited the expected 60000 molecular mass. The refolded PBP3 ' bound benzytpenicillin in a 1 to 1 molar ratio, was highly sensitive to aztreonam and showed the same degree of thermostability, in terms of penicillin-binding capacity, as the parent, membrane-bound PBP3, sug- gesting that protein refolding occurred with formation of the correct intramolecular interactions. Two to Received 18 March, 1988. 'Present address: Max-Planck Institui tiir ZiJchtungsfofschung, Kbin Vogelsang Egelopfad, D-5CIO0 Koln 30, FRG. fPresent address; Molecular Genetics Unit, Medical Research Council. Cambridge CB2 2QH, UK. ^Deceased (23 December, 1986). §Far corres- pondence. three mg of refolded PBP3** can be obtained from 1 litre of culture of the overproducing strain. Introduction The term 'active-site serine, penicillin-interactive protein or protein domain' applies to a whole set of bacterial enzymes: the p-lactamases of class A, C and D, the low-Mr DD-peptidases/penicillin-btnding proteins (PBPs), and the DD-peptidase/penicillin-binding domains of the high-M, PBPs (Joris et al., 1988). A common feature of these enzymes is reaction with penicillin and other p-lactam antibiotics in a 1 to 1 molar ratio via formation of a serine ester-linked acyl {penicilloyi, cephalosporoyi) enzyme. Gene sequencing has yielded the primary structure of about 20 of these enzymes (see Jorls ef al., 1988) and X-ray crystallography has revealed details of the three- dimensional structure and active-site environment of several p-lactamases of class A (Kelly et al.. 1986; Samraoui et al., 1986; Herzberg and Moult, 1987; Did- ebergefa/., 1987) and the naturally occurring, water-solu- ble, low-M, DD-peptidase/PBP of Streptomyces R61 (Kelly et ai. 1986). On the basis of these structural data, it has been proposed that, in all likelihood, all the penicillin-inter- active proteins or domains form a 'superfamily' of evolu- tionarily related enzymes (Joris etai, 1988). Although they have diverged greatly in terms of primary structure, and acquired varying specificities and functionalities, they would have conserved substantial similarities in their polypeptide 'scaffolding'. In spite of these advances, determination of the three- dimensional structure of the physiologically-important, hIgh-M, PBPs IA, IB, 2 and 3 of E. coli remains an important aim of the research in this field. As the results of biochemical and recombinant DNA experiments suggest, these high-M, PBPs contain both an amino terminal domain that catalyses a penicillin-insensitive peptido- glycan-transglycosylase reaction and a carboxy-terminal penicillin-binding domain that catalyses a penicillin-sensi- tive peptidoglycan transpeptidase reaction. With the exception of the aforementioned Streptomyces R61 DD- peptidase/PBP, the PBPs are membrane-bound. As a corollary, their crystallization must be achieved in the presence of detergent, an approach which so far has not yielded crystals suitable for X-ray diffraction analysis