Production of native creatine kinase B in insect cells using a baculovirus expression vector

Abstract

A full-length human creatine kinase B (B-CK) cDNA was used to produce a recombinant baculovirus (AcDZl-BCK). Sf9 cells infected with this recombinant expressed a homodimeric protein composed o f 43 kDa subunits which, under optimal condi tions, formed up to 30% o f the total soluble cellular protein. Upon analysis by PAGE, zymogram assay and gel filtration chro matography the recombinant protein behaved like authentic dimeric human BB-CK protein. Studies with a newly produced monoclonal antibody (CK-BYK/21E10) directed against an epitope in the N-terminus o f the protein confirmed the identity o f the product. The recombinant BB-CK protein was purified to over 99% homogeneity from the total protein extract o f A cD Zl-CKB infected cells in one single step involving anion exchange column chromatography on MonoQ in FPLC. Dialysed pro tein had a specific activity o f 239 U/mg protein. (Mol Cell Biochem 143: 59-65, 1995)

Mice lacking brain-type creatine kinase activity show defective thermoregulation

“…Protein samples were separated on a 10% SDS-page gel and transferred to nitrocellulose membranes (ECL, Amersham Pharmacia Biotech BA, Piscataway AB, Uppsla, Sweden) by western blotting. The membranes were blocked with 5% non-fat dry milk in PBST (PBS, 0.1% Tween-20) for 60 min and incubated overnight with primary mono-clonal anti-CK-B 21E10 (1:1000; [20]), polyclonal anti-UbCKmit #253 (1:1000; [21]), or polyclonal anti-UCP1 (1:5000, Chemicon International, Temecula, CA, USA) antibody at 4°C. The membranes were washed 3×5 min with PBST and incubated for 60 min with secondary peroxidase-conjugated Goat anti-Rabbit (Pierce Biotechnology Inc., Rockford, IL, USA) for use with UbCKmit and UCP1 antibodies, or ImmunoPure®Protein A/G (Pierce Biotechnology Inc) for use with CK-B 21E10 antibodies.…”

“…Sections were incubated in 0.375% H 2 O 2 and 0,1% Sodium Azide for 30 min at 37°C to inactivate endogenous peroxidase, rinsed in 0.01% PBS-Tween, and incubated for 30 min in blocking buffer containing 1% BSA and 2% normal donkey serum. Sections were incubated overnight at 4°C with polyclonal rabbit primary antibody for either CK-B (1:1000; [20]), or UbCKmit (1:1000; [21]). Next, sections were incubated for 1 h with a secondary biotinylated donkey anti-rabbit antibody (1:250 in PBS-T; Vector Laboratories, USA), and for 1 h with Avidine Biotinylated enzyme Complex (Vectastain Elite ABC kit, Vector Laboratories).…”

Co-localization and functional coupling of creatine kinase B and gastric H+/K+-ATPase on the apical membrane and the tubulovesicular system of parietal cells

“…Blots were incubated with specific antibodies for H+/K+-ATPase (a) and CK (b), and stained as described in the Materials and methods section. Controls are baculovirus-produced BB-CK (0.5 ,ug) [22] and H+/K+-ATPase (10 jig) [36]. CK activities (units/mg of protein) of all samples are indicated on top of each lane.…”

Local ATP Generation by Brain-Type Creatine Kinase (CK-B) Facilitates Cell Motility

“…Samples were resolved by 10% SDS-PAGE and blotted onto nitrocellulose membranes. Subsequently, the membranes were blocked with PBS containing 5% non-fat milk and blots were probed with anti-CK-B (21E10, 1∶2000) [34], polyclonal anti-CK-B, 1∶2000 [57] or anti-myc (1∶100, Developmental Studies Hybridoma Bank, University of Iowa) or anti-YFP, 1∶3000 [58], anti-GAPDH (1∶3000, Trevigen) or anti-Tubulin (1∶2000, Developmental Studies Hybridoma Bank, University of Iowa) for 1 hr at RT or overnight at 4°C. Antibodies were detected with HRP-conjugated goat-anti-mouse IgG (1∶10000, Jackson ImmunoResearch) and the signal was developed with the HRP substrate Lumi-Light system (Roche).…”