ABSTRACT
Insulin conjugates in which the B1Phe residue has been chemically modified often exhibit a reduced tendency to associate into hexamers due to weakened interactions between subunits. The purpose of this study was to prepare a hexamer formulation for such insulin conjugates by using Co(III) as a coordinating metal ion. PEGylated insulin in which monomethoxypoly(ethylene glycol) (mPEG, M(r) 5000 or 20,000) had been site-specifically attached to B1Phe was chosen as a model conjugate. Hexamerization of mPEG-insulin upon H(2)O(2)-mediated oxidation of Co(II) was kinetically and quantitatively analysed by visible spectrometry and size-exclusion HPLC. Co(III) mPEG-insulin hexamers thus obtained were extremely stable, existing mostly as a hexameric form even at nanomolar concentrations. A remarkable increase in hydrodynamic volumes was observed for Co(III) mPEG(20k)-insulin hexamers (1600 kDa), as well as Co(III) mPEG(5k)-insulin hexamers (300 kDa). Our results demonstrate the potential benefits of Co(III) hexamer formulation for weakly associating insulin conjugates in the treatment of diabetes.
Subject(s)
Cobalt/pharmacology , Insulin/analogs & derivatives , Insulin/metabolism , Protein Multimerization/drug effects , Calibration , Chromatography, High Pressure Liquid , Humans , Insulin/chemical synthesis , Insulin/isolation & purification , Molecular Weight , Polyethylene Glycols/chemical synthesis , Polyethylene Glycols/chemistry , Polyethylene Glycols/isolation & purification , Polyethylene Glycols/metabolism , Spectrum AnalysisABSTRACT
There are several conditions which might modulate polymerization to produce polymers having normal lattice structure. In the absence of 1 mM MgCl(2) the assembly was reduced by 36% in Capsicum annuum tubulin (CAnm tubulin). There was no significant difference in the final assembly formation in the presence of 5% to 10% glycerol. However, nucleation rate was slow and apparent study state was achieved lately in the presence of 10% glycerol. Taxol at 100 microM concentration increased 23% tubulin assembly. One millimolar CaCl(2), >or=1% dimethyl sulfoxide (DMSO) and physiologically low temperature reduced CAnm tubulin assembly. A value of 0.089 mg/ml was obtained as critical concentration for polymerization. Benomyl significantly reduced the number of cysteine residues accessible to 5,5'-dithiobis-(2-nitrobenzoic acid); there were 4.77 +/- 0.21 and 3.49 +/- 0.35 residues accessible per tubulin dimer in the presence of 50 and 100 microM benomyl respectively.
Subject(s)
Capsicum , Plant Proteins/chemistry , Plant Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Tubulin/metabolism , Dithionitrobenzoic Acid/metabolism , Indicators and Reagents/pharmacology , Protein Multimerization/drug effects , Protein Structure, Quaternary , Temperature , Tubulin/chemistryABSTRACT
Alpha and beta tubulin genes were cloned from the Capsicum annuum leaves using rapid amplification of cDNA ends (RACE)-PCR. Nucleotide sequence analysis revealed that 1,353 bp Capsicum annuum alpha/beta-tubulin (CAnm alpha/beta-TUB) encodes a protein of 450 amino acids (aa) each. The recombinant alpha/beta tubulin was overexpressed mainly as an inclusion body in Escherichia coli BL21 (DE3), upon induction with 0.2 mM isopropyl-beta-D-thiogalactopyranoside (IPTG), and its content was as high as 50% of the total protein content. Effective fusion protein purification and refolding are described. The average yields of alpha and beta tubulin were 2.0 and 1.3 mg/l of culture respectively. The apparent molecular weight of each tubulin was estimated to be 55 kDa by SDS-polyacrylamide gel electrophoresis (PAGE). The tubulin monomers were found to be assembly competent using a standard dimerization assay, and also retained antigenicity with anti-His/T7 antibodies. The purified tubulins were polymerized to microtubule-like structures in the presence of 2 mM guanosine 5'-triphosphate (GTP).