ABSTRACT
Protein disulfide isomerase-related protein A (PRPA) was highly expressed (about 34%) in Escherichia coli by inserting the whole PRPA cDNA into the vector pET23b. After expression, the purified protein was acquired through ammonium fractional precipitation and Bio-Rex 70 chromatography. PRPA shows low disulfide isomerase activity (only about 1/250 of that of hPDI), decreases the reactivation yield of denatured and reduced lysozyme either in redox and non-redox Hepes buffer or redox PBS buffer and facilitates the aggregation of denatured and reduced lysozyme. Fluorescence spectra of PRPA indicate that PRPA has more hydrophobic groups at surface than that of hPDI, and which can be used to explain why PRPA has anti-chaperone activity during the refolding of denatured and reduced lysozyme.
Subject(s)
Cloning, Molecular , Fungal Proteins , Chemistry , Genetics , Muramidase , Chemistry , Plasmids , Protein Folding , Recombinant Proteins , Spectrometry, FluorescenceABSTRACT
The template-independent teminal transferase activity of Taq DNA polymerase results in an overhanging dA at the 3′end of its PCR products. The pGEMX vector constructed in this experiment forms a single overhanging dT at its 3′end as the result of cleavage with Xcm I restriction enzyme. This vector is very efficient for direct cloning of PCR product obtained by using Taq DNA polymerase.Recombinant colonies can be selected by Blue/white screening. Moreover,insertion fragment can be easily released from the vector simply with either BamH I or Hind III digestion.