This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1（designated ΔSHP-1） and the preparation of its polyclonal antibodies. A cDNA fragment encoding ΔSHP-1 wa...This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1（designated ΔSHP-1） and the preparation of its polyclonal antibodies. A cDNA fragment encoding ΔSHP-1 was amplified by PCR and then cloned into the pT7 expression vector. The recombinant pT7-ΔSHP-1 plasmid was used to transform Rosetta（DE3） E. coli cells. ΔSHP-1 was distributed in the exclusion body of E. coli cell extracts and was purified through a two-column chromatographic procedure. The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns. It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases. To generate polyclonal anti-ΔSHP-1 antibodies, purified recombinant ΔSHP-1 was used to immunize a rabbit. The resultant anti-serum was subjected to purification on ΔSHP-1 antigen affinity chromatography. The purified polyclonal antibody displayed a high sensitivity and specificity toward ΔSHP-1. This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development.展开更多
A mixture of oligosaccharide and glycopeptide was isolated from the aqueous extract of Panax ginseng roots. The mixture inhibits protein tyrosinc phosphatase（SHP-1） function, implying it enhances immune activity. Th...A mixture of oligosaccharide and glycopeptide was isolated from the aqueous extract of Panax ginseng roots. The mixture inhibits protein tyrosinc phosphatase（SHP-1） function, implying it enhances immune activity. The peak molecular mass of the oligosaccharide portion is 1800 calculated via GPC software after separation by HPLC. And the structure of the oligosaccharide portion is the backbone of （1→3）- and （1→4）-linked arabinopyranoside, and （1→4）- and （1→6）-linked glucopyranoside, with non-reducing terminals of arabinopyranoside and glucopyranoside. The peak molecular mass of glycopeptide portion is 1900 calculated via GPC software after separation by HPLC. The structure of glycopeptide portion is the backbone of （1→3）- and （1→4）-linked arabinopyranoside, and （1→3,6）-linked glucopyranoside, with non-reducing terminals of galactopyranose and glucopyranoside. The peptide composition is Glu. Asp, Hyp, Set, Arg, Gly , Thr, Pro, Ala, Val, lie, Leu and Lys. The oligosaccharide-peptide linkage is formed by Ara and Hyp.展开更多
基金the Fund of Science & Technology Bureau of Jilin Province, China(Nos.20060563, 200705394 and 20080434).
文摘This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1（designated ΔSHP-1） and the preparation of its polyclonal antibodies. A cDNA fragment encoding ΔSHP-1 was amplified by PCR and then cloned into the pT7 expression vector. The recombinant pT7-ΔSHP-1 plasmid was used to transform Rosetta（DE3） E. coli cells. ΔSHP-1 was distributed in the exclusion body of E. coli cell extracts and was purified through a two-column chromatographic procedure. The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns. It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases. To generate polyclonal anti-ΔSHP-1 antibodies, purified recombinant ΔSHP-1 was used to immunize a rabbit. The resultant anti-serum was subjected to purification on ΔSHP-1 antigen affinity chromatography. The purified polyclonal antibody displayed a high sensitivity and specificity toward ΔSHP-1. This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development.
基金Supported by the National Science & Technology Pillar Program of China(No2007BA138B00)
文摘A mixture of oligosaccharide and glycopeptide was isolated from the aqueous extract of Panax ginseng roots. The mixture inhibits protein tyrosinc phosphatase（SHP-1） function, implying it enhances immune activity. The peak molecular mass of the oligosaccharide portion is 1800 calculated via GPC software after separation by HPLC. And the structure of the oligosaccharide portion is the backbone of （1→3）- and （1→4）-linked arabinopyranoside, and （1→4）- and （1→6）-linked glucopyranoside, with non-reducing terminals of arabinopyranoside and glucopyranoside. The peak molecular mass of glycopeptide portion is 1900 calculated via GPC software after separation by HPLC. The structure of glycopeptide portion is the backbone of （1→3）- and （1→4）-linked arabinopyranoside, and （1→3,6）-linked glucopyranoside, with non-reducing terminals of galactopyranose and glucopyranoside. The peptide composition is Glu. Asp, Hyp, Set, Arg, Gly , Thr, Pro, Ala, Val, lie, Leu and Lys. The oligosaccharide-peptide linkage is formed by Ara and Hyp.
