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Notes: The PinPoint™ Xa Protein Purification System was used to clone and produce recombinant human Dicer (re-hDicer). Blunt-ended cDNA coding hDicer was cloned into one of the PinPoint™ Xa vectors. Biotinylated re-hDicer was produced in E. coli by inducing cultures with 100μM IPTG in the presence of 2μM biotin. Recombinant hDicer was purified from E. coli lysates with SoftLink™ Soft Release Avidin Resin. The purified re-hDicer was demonstrated to have a putative molecular weight of ~220KDa. Recombinant-hDicer protein was also demonstrated to have RNase III-like activity by a processing assay using double-stranded puromycin resistance gene mRNA. (2706)

Notes: The the motor function of the DNA-binding kinesin-like protein Kid was studied. Biotinylated Kid domains were expressed using the PinPoint™ System and the motor force of  Kid pulling on an optically trapped streptavidin bead was detected.  (2703)

Notes: The authors used the PinPoint™ Xa-1 Vector to clone two herpes simplex virus (HSV) genes and express the encoded proteins, IE63 and thymidine kinase, in E. coli JM109 for use as immunogens. Proteins expressed from the PinPint™ Vectors have a biotinylated tag, and the authors used this tag to purify the HSV proteins using the SoftLink™ Soft Release Avidin Resin. In a large-scale purification, the typical yield of IE63 protein was ~650µg/L. The recommended PinPoint™ purification protocol was modified in several ways: 1) the optimal incubation temperature and time for protein induction in E. coli cultures was chosen based on JM109 growth curves; 2) the post-induction time was optimized by monitoring protein expression levels using Western blot analysis; 3) the length of time that proteins were allowed to bind to the SoftLink™ Resin was optimized. The authors found that shortening the length of binding time to 2 hours eliminated the co-purification of endogenous biotinylated proteins in E. coli. (3850)

Notes: The chURP protein was expressed from the PinPoint™ Xa-3 Vector in a 2-liter culture of JM109. Much detail is provided for purification of the protein to make an immunogen for antibody production. The Wizard^® Lambda Preps System was used to purify cDNA clones of the chURP and the Erase-a-Base^® System was used to make nested deletions for sequencing. (0741)

Notes: The PinPoint^® Xa System was used to produce recombinant Hantaan virus nucleocapsid proteins. The methods used in the production of the recombinant proteins is found in the following journal article: Yoshimatsu, K, et al. (1996) J. Gen. Virol. 77, 695-704. (0662)

Notes: The authors used the PinPoint™ Xa Protein Purification System to express the Grb2 SH3 domain for competitive inhibition studies of peptides that interfere with its interaction with the proline-rich domain of sos. (0607)

Notes: The human protein hsPex14p as welll as another protein hsPex13p and hsPex5p were cloned into the PinPoint^® Xa2 vector and expressed in JM109 E.coli. The cell extracts were run on SDS PAGE gels, transferred to nitrocellulose and probed with the anti-MF3 antisera. The hsPex5p, supposedly the human PTS1 homolog, was translated in vitro with the TNT^® Coupled Reticulocyte Lysate System and reacted with another blot with the same proteins and the hsPex14p bound the 35S-labeled protein. (1172)

Notes: Used PinPoint™ Xa-3 vector to express C-terminal segments of GluR6. (1139)

Notes: The authors used PinPoint™ Xa-1 Vector to express a biotinylated Pex5p protein for an in vitro ELISA-like assay. (0833)

Notes: The PinPoint™ Protein Purification System was used to express a 178 kDa protein. The protein was expressed, purified on the SoftLink™ Avidin and the purification tag cleaved with Factor Xa. (0563)

Notes: Eighty-five residue of CBP were inserted into the PinPoint™ Xa-1 Vector, expressed and purified on a monomeric avidin column. The purified protein was used to screen a human placenta cDNA expression library with the aid of streptavidin-alkaline phosphatase. Two clones were identified out of 3 million plaques. Both clones encoded the RNA helicase A. (0643)

Notes: The nine carboxyl terminal amino acids of NR2B, a subunit of the NMDA receptor, were fused into the PinPoint™ Xa3 Vector. The protein was expressed in DH5α and purified with the SoftLink™ Soft Release Avidin Resin. The purified protein was used to probe various bacterially-expressed fusion protein immobilized on nitrocellulose. Proteins containing the PDZ3 domain interacted with the biotinylated fusion protein and were visualized with a streptavidin-HRP conjugate. (0670)

Notes: The c-terminal 93 amino acids of ZnT-3 were expressed from a PinPoint™ Vector and the fusion protein was immobilized on an avidin column. The column-bound protein was used for affinity purification of rabbit immunoglobulins raised against the same peptide fused to maltose binding protein. (0580)

Notes: The 273 amino acid prtB cDNA was cloned into the PinPoint™ Xa-3 Vector and expressed in E. coli HB101 cells. The protein was purified using the SoftLink™ Soft Release Avidin Resin. The purified protein was treated with Factor Xa Protease. Both the fusion protein and the Factor Xa cleaved protease were functional enzymes. (1481)