当前位置： SCI文献检索 > MOLECULAR AND CELLULAR ENDOCRINOLOGY期刊下所有文献 > Single-column purification and bio-characterization of recombinant human parathyroid hormone-related protein (1-139).

Single-column purification and bio-characterization of recombinant human parathyroid hormone-related protein (1-139).

Abstract：

:Recombinant human parathyroid hormone-related protein (hPTHrP) (1-139) was expressed using the IMPACT T7 (intein-mediated purification with an affinity chitin-binding tag) system, allowing purification of free recombinant peptide in a single chromatographic step. This system utilizes an intein, which is a protein splicing element from the Saccharomyces cerevisiae VMA1 gene. The intein has been modified so that it undergoes a self-cleavage reaction at its N-terminus at low temperatures in the presence of 1,4-dithiothreitol (DTT). The cDNA encoding hPTHrP (1-139) was cloned into the pTYB1 vector to create an in-frame fusion at the N-terminus of the intein gene. The cDNA for the chitin-binding domain from Bacillus circulans is present at the C-terminus of intein for affinity purification of the three-part fusion protein on a chitin column. The recombinant plasmid was transfected into E. coli ER2566 cells and synthesis of the PTHrP fusion protein was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). This system produced pure hPTHrP (1-139) and an N-terminally truncated analogue, hPTHrP (27-139), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot analysis, N-terminal sequence analysis and mass spectroscopy. hPTHrP (1-139) stimulated cAMP accumulation in ROS 17/2.8 osteoblastic bone cells, whereas hPTHrP (27-139) failed to elicit a response. hPTHrP (1-139) also inhibited the growth of the breast cancer cell line MDA-MB-231; the magnitude of the response was comparable with that of synthetic hPTHrP (1-34) and (1-86). Neutralization of endogenous PTHrP and added hPTHrP (1-139) and N-terminal species with an anti-PTHrP antiserum completely abolished the growth inhibitory effects. These results indicate that the added peptides modulate cell growth by acting at the cell surface. Availability of recombinant hPTHrP (1-139) will allow further study of its biological function, as well as its structure.

journal_name

Mol Cell Endocrinol

journal_title

Molecular and cellular endocrinology

authors

Wu C,Seitz PK,Falzon M

doi

10.1016/s0303-7207(00)00323-3

keywords:

subject

Has Abstract

pub_date

2000-12-22 00:00:00

pages

163-74

issue

1-2

eissn

0303-7207

issn

1872-8057

pii

S0303-7207(00)00323-3

journal_volume

170

pub_type

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