Method to express and purify nm23-H2 protein from baculovirus-infected cells.
High-throughput protein expression and purification are major bottlenecks in the postgenomic and proteomic era. We show here an automated method to express and purify nm23-H2, a nucleoside diphosphate kinase (NDPK), in a 96-well format, by the use of a robotic workstation, from insect Spodoptera frugiperda (Sf9) baculovirus-infected cells using nickel-nitrilotriacetic acid (Ni-NTA) agarose beads. The automated method is coupled to mass spectrometry for a validation and quality-control analysis. To verify the bona fide of the recombinant protein, several tests have been produced, including NDPK assay, Western blotting, and in vitro phosphorylation experiments, thus confirming the value of the protocol developed. The method has been validated for the expression of several proteins, thus confirming the value of this automated protocol. The research presented here is a useful method both for industrial and academic environments to produce in a high-throughput mode recombinant eukaryotic proteins to be assayed for a specific function in a systematic manner.
Garzia
L
L
Telethon Institute of Genetics and Medicine, Naples, Italy.
André
A
A
Amoresano
A
A
D'Angelo
A
A
Martusciello
R
R
Cirulli
C
C
Tsurumi
T
T
Marino
G
G
Zollo
M
M
D000818:Animals; D016367:Baculoviridae; D015153:Blotting, Western; D047388:Casein Kinases; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D008970:Molecular Weight; D009154:Mutation; D054778:NM23 Nucleoside Diphosphate Kinases; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis; D015203:Reproducibility of Results; D012371:Robotics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera / Q000821:virology
D054778:NM23 Nucleoside Diphosphate Kinases; D011506:Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases; D047388:Casein Kinases; D009701:Nucleoside-Diphosphate Kinase