Cloning and expression of l-asparaginase from E. coli in eukaryotic expression system

• Asparaginase is an oncolytic agent which affects the growth of cancer cells.
• Pichia pink expression system has high expression rate.
• Cloning of Escherichia coli ansB gene in eukaryotic expression system.
• Increased activity and expression level.

l-Asparaginase is an anti-cancer agent which prevents the proliferation of cancerous cells by decreasing the level of asparagine in the blood. l-asparaginase from Escherichia coli which is encoded by ansB gene is widely used because of its substrate specificity and less glutaminase activity. Here, we are reporting the expression studies in yeast which has many added advantages like protein folding and processing. The expression studies were carried out in a new protein expression system based on the yeast Pichia pastoris called PichiaPink™. ansB gene isolated from E. coli which is coded for asparaginase was cloned into pPink HC-α plasmid and transformed into protease knock out pichia pink strain by electroporation. The recombinant enzyme was extracellular and showing the activity of 2.5 IU/ml. It was then purified using Ni-NTA column since the enzyme contains His-tag at the C-terminal end. The new way of expression would be efficient in making the enzyme humanized by glycosylation patterns which is similar to mammals.