Mutants of immunotoxin anti-Tac(dsFv)-PE38 with variable number of lysine residues as candidates for site-specific chemical modification. 1. Properties of mutant molecules.
Chemical modification of proteins with substances such as poly(ethylene glycol) can add useful properties to proteins. Currently PEGylation is done in a random manner utilizing amino residues dispersed throughout a protein. For proteins such as immunotoxins, which have several different functional domains, random modification leads to inactivation. To determine if we could produce an immunotoxin with a diminished number of lysine residues so that chemical modification could be restricted to certain regions of the protein, we chose the recombinant immunotoxin anti-Tac(dsFv)-PE38 that has 13 lysine residues in the Fv portion and 3 in the toxin. We prepared a series of mutants with 0-12 lysines in the Fv and 0 or 3 in the toxin. Almost all of these molecules retain full biological activity. Our data indicate that replacement of lysine residues can be achieve without loss of biological potency. These molecules are a useful starting point to carry out site-specific PEGylation experiments.
Onda
Masanori
M
Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Room 5106, Bethesda, Maryland 20892-4264, USA.
Vincent
James J
JJ
Lee
Byungkook
B
Pastan
Ira
I
D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity; D008239:Lysine / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D009841:Oligonucleotides / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013499:Surface Properties; D014407:Tumor Cells, Cultured
D004220:Disulfides; D000922:Immunotoxins; D009841:Oligonucleotides; D011506:Proteins; D011994:Recombinant Proteins; C474514:SS(dsFv)PE38; D008239:Lysine