L-Asparaginase from E. chrysanthemi expressed in glycoswitch^®: effect of His-Tag fusion on the extracellular expression

Abstract

L-Asparaginase (L-ASNase) is an important enzyme used to treat acute lymphoblastic leukemia, recombinantly produced in a prokaryotic expression system. Exploration of alternatives production systems like as extracellular expression in microorganisms generally recognized as safe (such as Pichia pastoris Glycoswitch^®) could be advantageous, in particular, if this system is able to produce homogeneous glycosylation. Here, we evaluated extracellular expression into Glycoswitch^® using two different strains constructions containing the asnB gene coding for Erwinia chrysanthemi L-ASNase (with and without His-tag), in order to find the best system for producing the extracellular and biologically active protein. When the His-tag was absent, both cell expression and protein secretion processes were considerably improved. Three-dimensional modeling of the protein suggests that additional structures (His-tag) could adversely affect native conformation and folding from L-ASNase and therefore the expression and cell secretion of this enzyme.

Keywords:

• L-Asparaginase
• extracellular expression
• Glycoswitch
• His-tag

Disclosure statement

The authors declare no competing financial interests.

Additional information

Funding

This research was supported by São Paulo Research Foundation (FAPESP/Brazil) (Grant numbers 2013/08617-7, 2015/07749-2, 2016/15787-4, 2017/20384-9, 2016/25896-5); Comisión Nacional de Investigación Científica y Tecnológica (CONICYT/Chile) (Grant number 21150288); DI12-PEO1 (EXE12-0004) DIUFRO. G. M. received a Productivity Fellowship from the Brazilian National Counsel of Technological and Scientific Development (CNPq 309595/2016-9).