High-level expression of three members of the murine angiogenin family in Escherichia coli and purification of the recombinant proteins.
Angiogenin (Ang) is a small basic protein which belongs to the pancreatic ribonuclease superfamily. It potently induces the formation of new blood vessels and has emerged as a promising anticancer target. Mice possess genes encoding one ortholog (mAng) and three homologs of Ang, designated angiogenin-related protein (mAngrp), angiogenin-3 (mAng-3), and angiogenin-4 (mAng-4). Structural and functional study of these homologs has been hampered by the low yield of protein from the existing heterologous expression system. In the experiments described, we used a pET expression vector to express these proteins in the cytoplasm of Escherichia coli BL21-CodonPlus(DE3)-RIL cells, whereupon substantial amounts of each accumulated in the form of insoluble aggregates. The proteins were renatured using an arginine-assisted procedure and subsequently purified by cation-exchange chromatography and reversed-phase HPLC; each purified protein was shown to be enzymatically active toward tRNA. The yields of pure mAngrp and mAng-3 were 7.6 and 12 mg/liter culture, respectively, representing substantial increases over previously reported experiments. This is also the first report of the expression and purification of mAng-4, obtained here in a yield of 30 mg/liter culture. The ready availability of milligram quantities of these proteins will enable further functional studies and high-resolution structural analyses to be conducted.
Holloway
D E
DE
Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom.
Hares
M C
MC
Shapiro
R
R
Subramanian
V
V
Acharya
K R
KR
D043925:Angiogenesis Inducing Agents / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D004926:Escherichia coli / Q000235:genetics*; D051379:Mice; D005810:Multigene Family; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D012259:Ribonuclease, Pancreatic / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D012260:Ribonucleases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
C403682:Ang3 protein, mouse; D043925:Angiogenesis Inducing Agents; D011506:Proteins; D011994:Recombinant Proteins; D012260:Ribonucleases; C046366:angiogenin; C099439:Ang2 protein, mouse; D012259:Ribonuclease, Pancreatic