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Title 

Use of carboxypeptidase Y propeptide as a fusion partner for expression of small polypeptides in Escherichia coli

Authors 

Gui Hwan OhMoon Sun HahmBong Hyun Chung

Publisher 

Elsevier

Issue Date 

1999

Citation 

Protein Expression & Purification, vol. 17, no. 3, pp. 428-434

Keywords 

affinity chromatographyamino terminal sequencecarboxypeptidase ccyanogen bromideenteropeptidaseEscherichia coliglucagonhybrid proteinliquid chromatographyparathyroid hormone

Abstract 

The carboxypeptidase Y (CPY) propeptide from Saccharomyces cerevisiae was developed as a fusion partner for the efficient expression of small polypeptides in Escherichia coli. Six consecutive histidine residues (6xHis) were fused to the N-terminus of the CPY propeptide for the facilitated purification of fusion proteins using immobilized metal ion affinity chromatography. In addition, a methionine or the pentapeptide (Asp)4-Lys linker was inserted at the junction between the CPY propeptide and the target polypeptide to release the target polypeptide by digestion with cyanogen bromide or enterokinase. Therapeutically valuable peptide hormones, such as salmon calcitonin precursor (sCAL-Gly), a fragment of human parathyroid hormone (hPTH(1-34)), and human glucagon were successfully expressed in E. coli as fusion polypeptides with the fusion partner. SDS-PAGE analyses showed that the majority of the expressed fusion sCAL-Gly and fusion hPTH(1-34) were present in the form of inclusion bodies, whereas about 66% of the expressed human glucagon was in a soluble form. Almost complete cleavage of the fusion polypeptides was obtained by digestion with enterokinase. Reverse-phase HPLC analyses showed that the target polypeptides released from the fusion proteins were identical to their native forms.

ISSN 

1046-5928

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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