Construction of expression systems for Escherichia coli asparaginase II and two-step purification of the recombinant enzyme from periplasmic extracts.
Isoenzyme II of Escherichia coli L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) is among the few enzymes of major therapeutic importance, being used in the treatment of acute lymphoblastic leukemia. We have constructed several inducible expression systems that overproduce asparaginase II from recombinant plasmids. The most efficient of these systems consists of plasmid pTWE1, a derivative of pT7-7, and an ansB- strain of E. coli, CU1783. These cells produce and secrete amounts of asparaginase II that account for 10-15% of the total cellular protein. Most of the active recombinant enzyme can be released from the periplasmic space by a simple osmotic shock procedure. From the resulting material homogeneous asparaginase II was obtained by a two-step procedure. Overall yields of purified asparaginase were 10-15 mg asparaginase II per liter of E. coli culture. The recombinant enzyme appeared identical to conventionally purified preparations.
Harms
E
E
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Wehner
A
A
Jennings
M P
MP
Pugh
K J
KJ
Beacham
I R
IR
Röhm
K H
KH
D000596:Amino Acids / Q000032:analysis; D001215:Asparaginase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence; D002458:Cell Fractionation; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009997:Osmotic Pressure; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D000596:Amino Acids; D004269:DNA, Bacterial; D007527:Isoenzymes; D011994:Recombinant Proteins; D001215:Asparaginase