Screening Cysteine Mutants for Site‐Specific PEGylation of L‐asparaginase II
Authors: Ramirez‐Paz Josell , Griebenow Kai 
Published: June 2021

L‐Asparaginase II is a therapeutically important enzyme that has been used for treating certain types of cancer since the 1970s, i.e., acute lymphoblastic leukemia, acute myeloid leukemia, and non‐Hodgkin's lymphoma. During chemotherapy, asparaginase is injected into the bloodstream to deplete the pool of circulating asparagine, an essential amino acid for lymphoblastic cells, which consequently affects protein biosynthesis in the malignant cells causing them to undergo apoptosis without affecting normal cells.[1,2] Some drawbacks limiting the efficiency of asparaginase are the rapid serum clearance and immune inactivation due to anti‐asparaginase antibodies developed by the host. This has been addressed by decorating the enzyme with biocompatible polymers. Specifically, a commercial formulation of E. coli L‐asparaginase II randomly conjugated with mono‐methoxy polyethylene glycol, reduced the incidence of neutralizing antibodies and exhibited longer blood half‐life activity than the non‐conjugated enzyme. Unfortunately, random modification also impacts negatively the drug's pharmacodynamics leading to drastically reduced biological activity (85–0%) and substrate affinity (KM).[3–6]In our previous work, we site‐selectively modified asparaginase with bi‐maleimide‐PEGs by linking the polymers to cysteines which were introduced by site‐specific mutagenesis. In addition, in order to reduce the number of polymers around the enzyme, we successfully cross‐linked the asparaginase subunits to generate a tetrameric and enzymatically active 1kDa‐PEG‐conjugate (~140 kDa by SDS‐Page) using a 1000 Da PEG polymers, and a higher molecular weight 5kDa‐PEG‐conjugate (~400 kDa by size exclusion chromatography) using 5000 Da PEGs. The last one exhibited superior enzymatic activity over the non‐conjugated asparaginase.[7] This novel idea represents a solution to increase the hydrodynamic size of the drug thus reducing glomerular filtration and affording prolong blood half‐life, while at the same time retaining full biological activity. However, this approach has a bottleneck which is the recombinant expression of asparaginase‐cysteine mutants. We observed that our double‐mutation (A38C‐T263C) reduces the expression of this variant to about 33% of the native recombinant asparaginase, tested in E. coli BL21(DE3) cells. In addition, purification was also challenging, since only 70% purity (by SDS‐Page) was achieved for this double‐mutant, in contrast to >95% for the native recombinant asparaginase.[7]To overcome these limitations, we designed a new expression system in which a His‐tag is introduced, along with deletion of the two natural Cys amino acids present in asparaginase. The last modification seeks to reduce undesired crosslinking during the PEGylation reaction, while the first one should easy the purification. We evaluated this system in terms of extracellular expression and final purity (after affinity chromatography). Our results show that this mutant can be purified relatively easy with high purity (>95% by SDS‐Page), but expression was almost inexistent. This let us to believe that the natural cysteine(s) in E. coli L‐asparaginase II play a key role in the extracellular secretion mechanism.[8]Support or Funding Information

Research facilities at the UPR ‐ Chemistry Department, Facundo Bueso
Research facilities at the UPR‐Molecular Sciences Research Center
RISE Program at the University of Puerto Rico ‐ Rio Piedras Campus
This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.