Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories.

Anil A. Sohail,Madhuri Gaikwad,Lloyd W. Ruddock,Prakash Khadka,Mirva J. Saaranen

doi:10.3390/ijms21030688

Abstract

Escherichia coli is the most widely used protein production host in academia and a major host for industrial protein production. However, recombinant production of eukaryotic proteins in prokaryotes has challenges. One of these is post-translational modifications, including native disulfide bond formation. Proteins containing disulfide bonds have traditionally been made by targeting to the periplasm or by in vitro refolding of proteins made as inclusion bodies. More recently, systems for the production of disulfide-containing proteins in the cytoplasm have been introduced. However, it is unclear if these systems have the capacity for the production of disulfide-rich eukaryotic proteins. To address this question, we tested the capacity of one such system to produce domain constructs, containing up to 44 disulfide bonds, of the mammalian extracellular matrix proteins mucin 2, alpha tectorin, and perlecan. All were successfully produced with purified yields up to 6.5 mg/L. The proteins were further analyzed using a variety of biophysical techniques including circular dichroism spectrometry, thermal stability assay, and mass spectrometry. These analyses indicated that the purified proteins are most likely correctly folded to their native state. This greatly extends the use of E. coli for the production of eukaryotic proteins for structural and functional studies.

Highlights

Protein production in both natural and recombinant systems can be broadly divided into two processes: synthesis of the polypeptide chain(s), and folding to the functional, native conformation
The successful production of these correctly folded proteins with the help of co- or pre-expression of Erv1p and protein disulfide isomerase (PDI) in the cytoplasm of E. coli, made us consider what the limitations might be in terms of the production of more complex disulfide bonded proteins
extracellular matrix (ECM) proteins are rich in disulfide bonds and they make good candidates to test the potential limitations of CyDisCo

Summary

Introduction

Protein production in both natural and recombinant systems can be broadly divided into two processes: synthesis of the polypeptide chain(s), and folding to the functional, native conformation. Disulfide bond formation is a complex process, but it can be split into two steps, de novo disulfide bond formation i.e., oxidation of dithiols to the disulfide state, and subsequent isomerization of non-native disulfides. The de novo reaction in the E. coli periplasm is catalyzed by DsbA/DsbB shuttle system while in eukaryotes it is catalyzed by enzymes from the sulfhydryl oxidase family e.g., Ero and Erv1p [5,6,7,8]. The isomerization reaction in the E. coli periplasm is catalyzed by DsbC with the aid of DsbD, while in the ER of eukaryotes it is catalyzed by members of the protein disulfide isomerase (PDI) family [5,7,8,9]

Methods

Results

Conclusion