Covalent Crosslinking of L‐Asparaginase II Subunits by Site‐Specific PEGylation

L‐Asparaginase II is a homo‐tetramer enzyme that catalyzes the hydrolysis of asparagine, which is an essential amino acid for leukemia cells. Used as a drug, asparaginase acts by depleting circulating asparagine after being injected into the blood, which consequently affects protein biosynthesis in the malignant cells causing them to undergo apoptosis without affecting normal cells. A commercial formulation of E. Coli asparaginase conjugated with monomethoxypolyethylene glycol (mPEG) has been successful when compared to the wild type enzyme in prolonging the half‐life (5–9 times) and reducing immune responses. This is mainly attributed to an increase in the molecular weight that prevents glomerular filtration and by a steric hindrance effect that hides antigenic epitopes on the enzyme's surface. On the other hand, random PEGylation reduces the enzymatic activity by more than 50% and inversely affects substrate affinity.1–5 A possible solution to this problem is to selectively attach the PEG‐polymers at specific residues on the enzyme. By this method, the mass of the enzyme can be increased while at the same time blocking specific antibody‐susceptible epitopes. So far, site‐specific PEGylation of asparaginase at canonical residues has not been reported. Our first approach was to covalently conjugate the asparaginase subunits through cysteine residues introduced by mutagenesis. In this manner, the mass of the active enzyme can be increased without the need to place an excess of polymers around each monomer. We expressed an active asparaginase mutant (A38C‐T263C) that allowed us to crosslinking the enzyme monomers using a simple thiol‐maleimide chemistry. We tested the conjugation with 1 kDa and 2 kDa maleimide‐PEG bivalent polymers. The 1 kDa neo‐conjugate rapidly formed a gel‐type material that to our surprise exhibited enzymatic activity. Furthermore, this asparaginase‐gel is reusable, which we tested by measuring the residual activity after thermal denaturation (20–80°C). We performed SEM to observe the texture of this gel. The 2 kDa neo‐conjugate remained soluble in buffer solutions (pH 7.4–8.6). We confirmed a high molecular weight for this neo‐conjugate similar to the native tetramer (~150 kDa) using native‐Page, SDS‐Page, and size exclusion chromatography. In addition, preliminary results suggest that the presence of cysteine residues is necessary for asparaginase secretion (to periplasm or culture medium) as a recombinant product. We performed a mutation (C77S‐C105S) to eliminate the only two natural cysteines in asparaginase. This mutation reduced the secreted expression to 5%, but the mutant‐enzyme exhibited 80% specific activity. Our future work focuses on performing site‐specific PEGylation but this time with 5, 10 and 20 kDa bi‐maleimide‐PEG polymer. Support or Funding Information Dr. Kai Griebenow's Applied Biochemistry and Biotechnology Laboratory at the University of Puerto Rico, Rio Piedras Campus. Research Facilities at the Molecular Sciences and Research Building at the University of Puerto Rico. Financial Support from the RISE Program at UPR‐Rio Piedras, NIH Grant No. 5R25GM061151‐14.