Expression in mammalian cells, purification and characterization of recombinant human pancreatic ribonuclease.
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 autopic 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.
Russo
N
N
Dipartimento di Chimica Organica e Biologica, Università di Napoli Federico II, Italy.
de Nigris
M
M
Ciardiello
A
A
Di Donato
A
A
D'Alessio
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012259:Ribonuclease, Pancreatic / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D011994:Recombinant Proteins; D012259:Ribonuclease, Pancreatic