Use of carboxypeptidase Y propeptide as a fusion partner for expression of small polypeptides in Escherichia coli.
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.
Oh
G H
GH
Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon, 305-600, Korea.
Hahm
M S
MS
Chung
B H
BH
D002116:Calcitonin / Q000235:genetics; D002268:Carboxypeptidases / Q000737:chemistry* / Q000235:genetics; D043402:Cathepsin A; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D005822:Genetic Vectors; D005934:Glucagon / Q000235:genetics; D006801:Humans; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000235:genetics; D011506:Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000737:chemistry*
D004792:Enzyme Precursors; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; C054296:parathyroid hormone-related protein (1-34); C028815:salmon calcitonin; D002116:Calcitonin; D005934:Glucagon; D002268:Carboxypeptidases; C468629:CTSA protein, human; D043402:Cathepsin A