Improved Production of Recombinant Myrosinase in Pichia pastoris.

Natália Andelová,Zuzana Rosenbergová,Miroslav Ferko,Martin Rebroš,Zuzana Hegyi

doi:10.3390/ijms222111889

Abstract

The effect of the deletion of a 57 bp native signal sequence, which transports the nascent protein through the endoplasmic reticulum membrane in plants, on improved AtTGG1 plant myrosinase production in Pichia pastoris was studied. Myrosinase was extracellularly produced in a 3-liter laboratory fermenter using α-mating factor as the secretion signal. After the deletion of the native signal sequence, both the specific productivity (164.8 U/L/h) and volumetric activity (27 U/mL) increased more than 40-fold compared to the expression of myrosinase containing its native signal sequence in combination with α-mating factor. The deletion of the native signal sequence resulted in slight changes in myrosinase properties: the optimum pH shifted from 6.5 to 7.0 and the maximal activating concentration of ascorbic acid increased from 1 mM to 1.5 mM. Kinetic parameters toward sinigrin were determined: 0.249 mM (Km) and 435.7 U/mg (Vmax). These results could be applied to the expression of other plant enzymes.

Highlights

Over the last two decades, proteins have become very important substances in many industrial, medical and research fields
The AtTGG1 myrosinase gene was expressed in P. pastoris in our previous study [13]
The analysis confirmed that the expressed gene contained an N-terminal native signal sequence encoding a 19-amino acid signal peptide

Summary

Introduction

Over the last two decades, proteins have become very important substances in many industrial, medical and research fields. Recombinant production of proteins is often used to produce large amounts of proteins needed for industrial and pharmaceutical applications. One of the most widely used hosts for protein expression is the Gram-negative bacterium Escherichia coli, due to its rapid multiplication, inexpensive nutritional requirements, fast and easy transformation, and high-level expression of the recombinant protein. The frequent misfolding and aggregation of recombinant proteins is one of the main disadvantages of this expression host [2]. This expression system cannot be used for the production of proteins requiring posttranslational modifications (e.g., glycosylation), which are crucial for the correct folding and activity of eukaryotic proteins

Methods

Results

Conclusion