Amino-acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells useful for NMR studies.
Culture conditions for successful amino-acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells are described. The method was applied to the selective labeling of the catalytic domain of c-Abl kinase with (15)N-phenylalanine, (15)N-glycine, (15)N-tyrosine or (15)N-valine. For the essential amino acids phenylalanine, tyrosine and valine high (15)N-label incorporation rates of >/=90% and approximately the expected number of resonances in the HSQC spectra were observed, which was not the case for the non-essential amino acid glycine. The method should be applicable to amino-acid-type selective isotope labeling of other recombinant proteins which have not been amenable to NMR analysis.
Strauss
André
A
Novartis Pharma AG, Central Technologies, Oncology Research, CH-4002 Basel, Switzerland. andre.strauss@pharma.novartis.com
Bitsch
Francis
F
Cutting
Brian
B
Fendrich
Gabriele
G
Graff
Patrick
P
Liebetanz
Janis
J
Zurini
Mauro
M
Jahnke
Wolfgang
W
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D020134:Catalytic Domain; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured / Q000378:metabolism / Q000821:virology; D003001:Cloning, Molecular; D016313:Genes, abl; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D016315:Proto-Oncogene Proteins c-abl / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology
D000596:Amino Acids; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins; D016315:Proto-Oncogene Proteins c-abl