Construction of phosphorylatable chimeric monoclonal antibody CC49.

Construction of Phosphorylatable Chimeric Monoclonal Antibody CC49 with a Casein Kinase I Recognition Site

“…The expression vector pdHL7 is a modification of the expression vector pdHL2 (27) containing the enhancer and promoter of CMV in place of the metallothioneine promoter and IgH-chain enhancer in both the L chain and H chain transcription units. The new vector, pdHL7, also provides for a 3Ј-untranslated region and polyadenylation site from the mouse C region at the end of the modified H-chain as described earlier (24,28). Escherichia coli strain DH5␣-competent cells were prepared as described (29).…”

Introduction of Protein Kinase Recognition Sites into Proteins: A Review of Their Preparation, Advantages, and Applications

“…32 P than 125 I, the specific radioactivity of 32 P-labeled proteins is over fourfold that of 125 I-labeled proteins per incorporated radioisotope moiety. Furthermore, multiple kinase recognition sites can be incorporated into proteins without altering the activity of the molecule (5), whereas this is difficult with iodination since the greater the number of random sites of covalent modification, the greater the inactivation of the proteins. Nonradioactive phosphate ( 31 P) can also be incorporated into these proteins to permit the detection of the phosphoserine and phosphotyrosine derivatives sensitively by Western blotting with antibodies against phosphoserine and phosphotyrosine residues, respectively, in the intact proteins or by techniques such as mass spectroscopy.…”

“…The MAb-chCC49-6P is labeled with [␥-32 P]ATP and the cAMP-dependent protein kinase as described previously (5). Approximately 10 g of MAb is incubated at 30°C for 60 min with 0.5 mCi of [␥-32 P]ATP (sp act 6000 Ci/mmol; Du Pont-New England Nuclear) and 25 units of the catalytic subunit of cAMP-dependent protein kinase from bovine heart muscle (specific activity of Ն20,000 units/mg, from Sigma; dissolved in 6 mg/ml DTT) in 25 l of 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 12 mM MgCl 2 , then cooled on ice to stop the reaction.…”

“…32 P]MAb-chCC49K1 and [ 32 P]MAbchCC49-6P to MCF-7 4C10 human breast carcinoma cells was performed as described (5). MCF-7 4C10 cells used for binding studies were grown to confluence in 6-well tissue culture plates in medium (GIBCO DMEM) supplemented with 10% fetal bovine serum, 0.05 mg/ml sodium pyruvate, 0.005 mg/ml insulin, 0.5ϫ nonessential amino acids and treated with 1000 units/ml Hu-IFN-␥ for 48 h before assay.…”

“…Fusions were constructed with a variety of proteins and various phosphorylation sites (3,(5)(6)(7)(8)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28): interferon (3), monoclonal antibodies (5-8), retinoblastoma protein (15,19), osteopontin (16), c-Fos (17), calmodulin (18), diphtheria toxin (20), endothelial growth factors (20), enterotoxins (20), lymphokines (20), ricin (20,21), antibody fragments (23), microtubule-associated protein (25), Escherichia coli RNA polymerase ␤Ј and subunits (27,28), IL-3 (C. Miyamoto, personal communication), IFN-␤ (X.-X. Zhou, J. Langer, and S. Pestka, unpublished data), and IL-10 and vIL-10 (L. Izotova, S. Kotenko, S. Saccani, and S. Pestka, unpublished observations).…”