Production of engineered human pancreatic ribonucleases, solving expression and purification problems, and enhancing thermostability.
Human pancreatic ribonuclease, the homolog of bovine pancreatic ribonuclease, has a significant therapeutic potential. Its study has been hindered by the difficulty of obtaining the enzyme in a pure and homogeneous form, either from human source or using heterologous expression. Engineering of different variants of human pancreatic ribonuclease has allowed us to study and overcome some problems encountered during its heterologous production in an Escherichia coli system and its purification from inclusion bodies. The 5'-end region of the mRNA that encodes the enzyme is critical for obtaining high expression levels. The results also suggest the importance of the proline 50 residue in the recovery yields of human pancreatic ribonuclease. All the variants produced are pure and homogeneous. Their homogeneity has been demonstrated by cation-exchange and reversed-phase chromatography and by mass spectrometry analysis. Moreover, enhancement of human pancreatic ribonuclease thermal stability is observed when residues R4, K6, Q9, D16, and S17 are changed to the corresponding residues of bovine seminal ribonuclease.
Canals
A
A
Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi, Girona, 17071, Spain.
Ribó
M
M
Benito
A
A
Bosch
M
M
Mombelli
E
E
Vilanova
M
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010179:Pancreas / Q000201:enzymology*; D015202:Protein Engineering; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D012661:Semen / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D012333:RNA, Messenger; D011994:Recombinant Proteins; D012259:Ribonuclease, Pancreatic