In this report, we present DNAzol, a patent-pending DNA isolation reagent containing guanidine thiocyanate and a detergent mixture. It is a complete, nontoxic and ready-to-use reagent for the isolation of genomic DNA from various biological sources. In the DNAzol protocol, a biological sample is homogenized (or lysed) in DNAzol, and the DNA is precipitated with ethanol, washed and dissolved in 8 mM NaOH. Following pH adjustment, the DNA can be used immediately for analysis or stored at 4 degrees C. The entire isolation can be completed in 20-30 min, and a wide range of DNA molecules can be isolated including genomic DNA and DNA fragments down to 0.1 kb in length. If necessary, samples can be stored in DNAzol at room temperature for extended periods of time. The isolated DNA is ready for PCR, Southern blotting and other molecular biology applications without any additional purification.

Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids Lys 2-Arg-1 and removal of a Lys'56-Arg'57 dipeptide connecting the light and heavy chains. This processing was inefficient in the mammary gland of transgenic mice and pigs. We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes. We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland. In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites. These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity.

No abstract

We have previously described the expression of the human proprotein convertase furin or paired basic amino acid-cleaving enzyme, in mice transgenic for paired basic amino acid-cleaving enzyme and human Protein C (HPC). Here we show 100-fold or higher expression of furin in the mammary gland, compared with endogenous furin. Furin and recombinant HPC were detected in the same regions of the mammary gland and regulated similar to the endogenous whey acidic protein. In addition to the expected intracellular localization, furin was secreted into the milk as an 80-kDa form lacking the transmembrane and cytoplasmic domains. Furin present at levels of up to 40,000 units/ml milk cleaved the t-butoxycarbonyl-RVRR-AMC substrate with a K m of 32 M, and processed the recombinant HPC precursor at the appropriate sites. Surprisingly, the expression of an active protease was not toxic to the mammary gland. This is a rare example of an animal model secreting active truncated forms of a processing endoprotease into a bodily fluid.Furin or paired basic amino acid cleaving enzyme (PACE) 1 is a Ca 2ϩ -dependent serine protease that processes proteins in the constitutive secretory pathway (1). Furin belongs to a family of subtilisin-like proprotein convertases including kexin (EC 3.4.21.61), PC1/3, PC2, PC4, PC5/6, PACE4 (reviewed in Ref. 1), and PC7/PC8 or lymphoma proprotein convertase (2-5), that play a role in the maturation of proproteins by cleavage at dibasic or tetrabasic sites (6). Unlike PC1/3, PC2, and PC4, which are restricted to endocrine, neural, or testicular germ cells and ovaries, respectively, the furin gene is expressed in most cells and tissues examined (1, 7).cDNAs cloned from several species show furin to contain prepro-, catalytic and middle domains, a cysteine-rich region, transmembrane anchor, and cytoplasmic domain (1). Furin is activated by autocatalytic cleavage of its propeptide, presumably at the -Lys-Arg-Arg-Thr-Lys-Arg Ϫ1 -2 site (1,8). A membrane-associated protein, it is found mainly in the trans-Golgi network (TGN) co-localized with the TGN 38 marker protein (9). Some amount of furin recycles between the cell surface and the TGN (9) in endosome-like structures (10). So far furin has not been detected in bodily fluids like blood, nor has the effect of its expression on cell and organ development been studied in transgenic animals.Transgenic animals secreting recombinant proteins into milk, blood, urine, or saliva (11) have been generated and differences in recombinant protein modification compared with the human counterpart observed (12). In a pioneering attempt to engineer the post-translational capacity of the mouse mammary gland, we reported enhanced proteolytic maturation of the recombinant human Protein C (rHPC) precursor upon coexpression with furin (13). However, the effect of increased furin concentration on mammary gland development could not be predicted, nor its' intra-and extracellular distribution. Here we demonstrate localization of two heterologous proteins with the endogenous whey ac...

No abstract