Production of mature bovine pancreatic ribonuclease in Escherichia coli.
The coding sequence for the bovine pancreatic ribonuclease (RNase) precursor has been cloned and produced in Escherichia coli using the polymerase chain reaction (PCR) technique. A PCR amplification has been carried out utilizing as template the recombinant plasmid, pQR138, which contains the coding sequence for the RNase precursor, and primers that allow for the addition of new sequences at the 5' and 3' ends of the coding sequence. The resultant fragment contains two coding sequences, one for a hexapeptide and the other for pre-RNase. This fragment has been cloned into the expression vector, pKK223.3, under the control of the tac promoter, to form a two-cistron vector. Upon induction with IPTG, E. coli cells harboring this construct generate a bicistronic mRNA which upon translation produces a hexapeptide and pre-RNase. The RNase precursor is efficiently translocated into the periplasmic space of E. coli. Upon translocation, the signal sequence is removed generating mature RNase. Formation of the disulfide bridges in RNase is facilitated by the oxidative environment of the periplasm and a fully active protein is obtained. RNase produced in E. coli has been purified to homogeneity by cation-exchange chromatography, and the removal of the signal sequence has been verified by N-terminal sequencing. The total process from inoculation of media to obtaining pure and fully active recombinant RNase is achieved in 48 h.
Tarragona-Fiol
A
A
Department of Biochemistry and Molecular Biology, University College London, UK.
Taylorson
C J
CJ
Ward
J M
JM
Rabin
B R
BR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012260:Ribonucleases / Q000235:genetics* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D012260:Ribonucleases