Cloning and expression of recombinant human arylsulfatase b in pichia pastoris

Nahid Sedighi khavidak,1 Fataneh fatemi,2,* Seyed omid ranaei siadat,3 Sareh arjmand,4

1. Shahid Beheshti University
2. Shahid Beheshti University
3. Shahid Beheshti University
4. Shahid Beheshti University

Abstract

Introduction

Arylsulfatase b belongs to the sulfatase family (arsb; n-acetylgalactosamine-4-sulfatase) (ec 3.1.6.12), is the lysosomal enzyme that removes the 4-sulfate group of n-acetylgalactosamine-4-sulfate at the non-reducing end of chondroitin-4-sulfate (c4s) and dermatan sulfate (ds) and thereby regulates their degradation. the lack of arsb associated with mucopolysaccharidosis vi (maroteaux–lamy syndrome) and leads to the accumulation of its substrate, dermatan sulfate, and chondroitin 4-sulfate, which results in severe skeletal abnormalities, cardiac valve disease, reduced pulmonary function, and other malfunctions including the cns involvement. while there is no currently approved treatment for mps vi, enzyme replacement therapy (ert) using human recombinant arsb became available recently. so far, the human recombinant arsb is produced in mammalian expression system which is a high cost and time-consuming process. the yeast expression system has considered as the suitable host for the expression of many mammalian genes due to the specific characteristics and low-cost recombinant protein production process. the aim of this study was to produce the recombinant human arsb in the methylotrophic yeast pichia pastoris.

Methods

Cloning: native human arsb and the human formylglycine-generating enzyme (sumf1) cdna with a signal peptide coding sequence, was inserted in ppic9 plasmid to produce the expression vector ppic9-arsb-sumf1. vectors were linearized and used to transform competent cells of p. pastoris, x33 and x33 humanized strain. an empty ppic9 vector was used as a negative control. gene insertion was confirmed by pcr and sequencing. expression: p. pastoris clones were grown in ypd medium (yeast extract 1% p/v; peptone 2% p/v; dextrose 2% p/v) during 48 hours at 28 °c and 220 rpm. cells were harvested by centrifugation and the pellet was resuspended in bmg medium and cultured for 24 hours at 28°c and 220 rpm. finally, cells were recovered and resuspended in bmm medium and cultured for 120 hours at 28 °c and 250 rpm. methanol was added every 24 h to maintain a final concentration of 0.5%. arsb expression verified using sds-page and western-blot.

Results

The results of pcr, double digest and sequencing indicated that the human arsb and sumf1genes were inserted successfully in ppic9 plasmid. the analysis of transformed colonies compare to the negative control, showed the presence of expression vector ppic9-arsb-sumf1in the x33 and x33 humanized p. pastoris’s strain. evaluation of 25ml scale of p. pastoris culture for human arsb-sumf1 showed the presence of arsb activity for the intracellular and extracellular fraction.

Conclusion

Human arylsulfatase b and formylglycine-generating enzyme genes were cloned successfully in p.pastoris x33 and x33-humanized strain. the presence of enzyme activity in the intracellular and extracellular fraction of cell, indicated that despite of using signal peptide the recombinant arsb was not secreted completely to the medium. nevertheless, the condition of arsb expression in p.pastoris should be optimized.

Keywords

Mucopolysaccharidosis vi, arylsulfatase b, formylglycine-generating enzyme, pichia pastoris