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Bovine seminal RNAase (BS-RNAase) is a singular ribonuclease of the pancreatic RNAase superfamily, with unusual structural, catalytic, and biological properties [1]. It is the only dimeric protein in the superfamily, with intersubunit disulfide bridges [2]. In native BS-RNAase the majority of the subunits interchange their N-terminal segments, while in the remaining dimers each N-terminal segment folds onto its respective subunit [3]. It is homotropically regulated in the second, rate determininig step of the catalytic reaction, with negative and positive cooperativity [4]. It is selectively deamidated; this produces a characteristic isoenzymic composition, made up of α2, αs and s2 isoenzymes, produced by the progressive transformation of β subunits into α subunits through selective deamidation at Asn67 [5]. It is a RISBASE, i.e. a Ribonuclease with Special Biological Actions, including its antitumor, anti-spermatogenic and immunosuppressive actions [1]. All these properties are lost when the enzyme is monomerized, although the monomeric derivative is fully and catalytically active [1,4]. A definition at the molecular level of the structural determinants underlying these diverse biological properties may help to shed light on the molecular mechanisms of such activities. To reach this goal, protein engineering can be a very effective approach, as it should allow to verify the effects of appropriate mutations on the catalytic and the “special” biological activities of the enzyme.

A PCR procedure for synthesizing genes which uses medium lenght oligonucleotides is described. The procedure is tested leading to the synthesis of the cDNA coding for HL-RNase.

A chimeric cDNA was constructed, coding for the N-terminal region of BSRNase (residues 1-49) and the C-terminal region of RNase A (residues 50-124). The resulting chimeric DNA was expressed in Escherichia coli and found to code for RNase chains that spontaneously assembled into a covalently dimeric ribonuclease.

Bovine seminal ribonuclease, a uniquely dimeric pancreatic-like RNase, with its dimeric structure stabilized by two intersubunit disulfides, and endowed with special, i.e. non-catalytic, actions (antitumor, immunosuppressive, antispermatogenic), was stably expressed in Chinese hamster ovary cells. The recombinant protein, secreted in the culture medium as a correctly folded dimeric enzyme, was purified to homogeneity and found to be fully active both in its catalytic and antitumor activities. © 1993.

Bovine seminal RNAase (BS-RNAase), an unusually dimeric member of the pancreatic-like ribonuclease superfamily, is also a multifunctional biological effector, with antitumor, immunosuppressive, and antispermatogenic activities. We report here the cloning of a semi-synthetic cDNA coding for the protein subunit chain, its expression with a T7 expression system in Escherichia coli inclusion bodies, the dimerization of correctly reoxidized monomeric protein, followed by the purification in high yields of the recombinant enzyme, and by its conversion to a protein undistinguishable from BS-RNAase as isolated from seminal vesicles, both in its catalytic activity and in the micro-heterogeneity of its quaternary structure. © 1993 Academic Press, Inc.

A synthetic cDNA coding for human pancreatic RNase, equipped with a secretion signal sequence, was cloned and stably expressed in Chinese hamster ovary cells. The recombinant RNase, secreted into the culture medium, was purified and characterized. It was found to be indistinguishable, by structural and catalytic parameters, from the enzyme isolated from human pancreas. Furthermore, the glycosylated forms were separated from the non-glycosylated form. Up until now, human RNases have been isolated only in small amounts from autoptic specimens. This has hindered the exploitation of a human RNase for the construction of immunotolerated immunotoxins. On the other hand, the availability of an effective system for the expression of a human RNase may render feasible the transfer, by protein engineering, of the interesting pharmacological actions of non-human RNase [1993 Trends Cell Biol. 3, 106-109] to an immunotolerated, human RNase.