Recombinant Heparinase (r-Heparinase). r-Heparinase was expressed using the pET system (21), where expression is driven by bacteriophage T7 polymerase. The host E. coli strain BL21(DE3) contains a chromosomal copy of the T7 polymerase gene under the control of lacUV5. The expression is induced by isopropyl f3-D-thiogalactoside. Two constructs were designed to express heparinase in the pET system. The first construct included the native heparinase leader sequence. The second construct started with a sequence that read Met, Gln22, Gln23, Lys24, Lys2s, Ser26 ....
The Met residue was added before the Gln22 to introduce a start codon. Nde I and BamHI restriction sites were appended onto the 5' ends of the N- and C-terminal primers, respectively. F. heparinum genomic DNA was used as a template for 10 scaled-up PCR cycles (see Amplification of the PCR Product) to generate the modified heparinase I product. The PCR product was isolated as discussed above and concentrated using a Centricon P-100 (Amicon) at 5000 x g for 20 min. The amplification product was treated with Klenow fragment of DNA polymerase I (New England Biolabs) for 15 min and with T4 DNA polymerase (New England
Biolabs) for 10 min. The PCR product was isolated on an agarose gel as described above. The blunt PCR fragment was concentrated using a Centricon P-100 and ligated with 20 ng of Sma I-digested pUC18 (Pharmacia) (19). The ligation mixture was then used to transform DH5a-competent cells (GIBCO/BRL/Life Technologies), as recommended by the manufacturer. Subcloned heparinase PCR fragments were excised from pUC18 by digestion with Nde I and BamHI, gel purified, and then ligated to pET-3a plasmid (predigested at the Nde I and BamHI sites and gel purified) using T4 DNA ligase (New England Biolabs). The ligation mixture then was used to transform DHSa-competent cells. The plasmid containing the heparinase I gene in pET-3a was isolated, purified, and used to transform the host cell BL21(DE3) (Novogen,
Madison, WI).
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提示 heparinase:肝素酶 gel:凝胶 plasmid:质粒
recombinant 重组子 host:宿主
机器翻译的就不用发上来了,浪废资源...