ABSTRACT
AX15 is a mutein of naturally occurring human ciliary neurophic factor (hCNTF), with improved biological activity, stability and solubility. AX15 is susceptible to protease degradation when expressed in Pichia pastoris. Amino acid sequencing revealed the degradation was occurred behind position 12 and 13 amino acid residues, which constitute a dibasic site, RR. Based on the substrate specificity of KEX2, a KEX2 resistant mutein of AX15-AX15 (R13K) was constructed, in which RR was replaced by RK. It was demonstrated that the stability of AX15 (R13K) improved significantly, as no degradation was detected even after 120 hours of induction. AX15 (R13K) was purified to homogeneity by ultrafiltration and gel filtration. TF-1 cell survival bioassay showed AX15 (R13K) had equivalent specific activity to AX15. The protease resistant mutein of AX15 may have greater in vivo stability and thus have superior therapeutic potential.
Subject(s)
Humans , Ciliary Neurotrophic Factor , Genetics , Genetic Vectors , Mutant Proteins , Genetics , Mutation , Peptide Hydrolases , Chemistry , Pichia , Genetics , Metabolism , Recombinant Proteins , GeneticsABSTRACT
Human parathyroid hormone (hPTH) was highly expressed in Escherichia coli by inserted the synthesized whole hPTH cDNA into the vectors pBV220 and pET22b. After expression and disruption, the purified product was acquired through cation exchange chromatography and reverse phase chromatography. From the results of N-terminal sequencing and MALDI-TOF-MS analysis the recombiant prtein was indentified as intact hPTH. In in vitro Bioassays the recombinant hPTH stimulated adenylate cyclase as the standard did. In ovariectomized rats the recombinant hPTH markedly increased the femoral bone mass and bone mineral density.
Subject(s)
Animals , Female , Humans , Rats , Amino Acid Sequence , Base Sequence , Bone Density , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Genetics , Metabolism , Molecular Sequence Data , Ovariectomy , Parathyroid Hormone , Chemistry , Genetics , Metabolism , Pharmacology , Rats, Wistar , Sequence Alignment , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The 5' region of the cell wall protein(CWP) gene containing multiple tandem promoters and the signal peptide-coding sequence was isolated by PCR from Br. brevis 50, and used to construct the shuttle vector pBKE50, which included the replication origin of pUB110 and the erythromycin-resistance gene of pGK12. The alpha-amylase gene of Bacillus subtilis 168 was ligated to pBKE50, producing plasmid pBKE50/alpha-amy. After the resulting plasmid was introduced into Br. brevis 50, soluble and biologically active alpha-amylase was secreted directly into the culture medium. The expression level of alpha-amylase in the recombinant Br. brevis 50 was twice higher than that of the donor strain.