Efficient production of biologically active human salivary cystatins in Escherichia coli.
Different Escherichia coli expression systems were used for expression of cDNA clones encoding the human salivary cysteine proteinase (CysP) inhibitors, cystatins SN and S (CsnSN and CsnS). These included pOTSNco12 that expresses foreign sequences as authentic (nonfusion) proteins, and pGEX-2T that directs the synthesis of foreign polypeptides as fusion proteins with glutathione S-transferase (GST). The pOTS vector produced low levels of recombinant CsnSN (reCsnSN) that was localized in the soluble fraction, but not easily purified. The pGEX vector, on the other hand, produced much higher yields of the fusion protein, GST::CsnSN, that was localized almost entirely in the insoluble protein fraction. Solubilized and refolded GST::CsnSN inhibited the CysP, papain, more efficiently than chicken egg white Csn, indicating that the recombinant product was biologically active and that the GST carrier did not interfere with the biological activity. The pGEX-2T vector was subsequently used for the large-scale production of reCsnSN and reCsnS that were cleaved from the GST by thrombin and purified by DE-52 cellulose chromatography. ReCsnSN inhibited papain almost as efficiently as salivary CsnSN, while the reCsnS showed lower inhibitory activity as compared to both salivary CsnS and reCsnSN.
Bobek
L A
LA
Department of Oral Biology, School of Dental Medicine, State University of New York, Buffalo 14214.
Wang
X
X
Levine
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D015891:Cystatins / Q000096:biosynthesis* / Q000302:isolation & purification; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010206:Papain / Q000037:antagonists & inhibitors; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D055333:Salivary Cystatins
D015891:Cystatins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D055333:Salivary Cystatins; D010206:Papain