In-vitro Cleavage of a Fusion Protein Bound to Cellulose Using the Soluble YscFs (Kex2) Variant

Overview

Journal Appl Microbiol Biotechnol

Specialties Biomedical Engineering
Biotechnology
Microbiology

Date 1992 Aug 1

PMID 1368916

Citations 1

Authors

P G Seeboth

R A Warren

J Heim

Affiliations

Soon will be listed here.

Abstract

In order to show site-specific cleavage of fusion proteins with an engineered soluble yscF variant, we have constructed a fusion gene encoding eglinC from Hirudo medicinalis and the cellulose-binding domain from the Cellulomonas fimi exoglucanase (Cex). The two fusion partners were separated by a Lys-Arg-containing recognition sequence for the yeast endoprotease yscF (Kex2). The fusion protein (eglinC-Cex) was expressed intracellularly in Saccharomyces cerevisiae. After disruption of the cells eglin-C-Cex was shown to bind to cellulose when present in total crude cell lysates. This step efficiently removed the majority of contaminating host proteins. While immobilized on cellulose, eglin-C-Cex could be cleaved into the expected fragments. Upon cleavage the eglinC part was released from the cellulose, while the purification tag, i.e. the cellulose binding domain, stayed bound to the cellulose matrix.

Citing Articles

Production of autolysis-proof Kex2 protease from Candida albicans in Saccharomyces cerevisiae for in vitro processing of fusion proteins.

Kim M, Sung B, Kim H, Sohn J, Bae J Appl Microbiol Biotechnol. 2022; 106(21):7063-7072.

PMID: 36195703 DOI: 10.1007/s00253-022-12212-6.

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