Fig 1.
Selection of L-asparaginase PEGylation sites.
(A) L-Asparaginase tetramer with subunits highlighted with different colors, one active site is represented in red spheres, the pre-selected PEGylation positions in blue spheres, and the distances between mutated residues (A38C and T263C) are shown by black dotted lines. (B) L-Asparaginase dimer showing two active sites relatively buried (red spheres) in comparison with the PEGylations sites (blue spheres).
Fig 2.
Cysteine-directed PEGylation crosslinking with 1 and 2 kDa PEG.
(A) 1kDa-PEG-conjugate gel. (B) Native and (C) SDS-PAGE of native L-asparaginase (left lanes) and the 2kDa-PEG-conjugate (right lanes). Note that a substantial amount of the 2kDa-PEG-conjugate did not enter the gel because it was too large. The bio-PEG-conjugates were generated by the ultrafiltration crosslinking method and afterward purified with one gel filtration step. The photo of the 1kDa-PEG-conjugate gel was taken on a blue light dock, the hydrogel had a clear appearance.
Fig 3.
Cysteine-directed PEGylation followed by size exclusion chromatography.
(A) Mutant L-asparaginase (A38C-T263C) reduced with TCEP and eluted from the gel filtration column. Fractions 8–13 ml were pooled and used for the PEGylation crosslinking reaction. (B) 5kDa-PEG-Conjugate after crosslinking reaction, reduced with TCEP and eluted from the gel filtration column. The continuous curve represents the relative absorbance at 280 nm and the bars are the relative asparaginase catalytic activity of the fractions.
Table 1.
Catalytic activity of cysteine-directed crosslinked L-asparaginase.
Table 2.
Comparison of PEGylation methods for L-asparaginase.
Fig 4.
Dependence of L-asparaginase catalytic activity on the degree of PEGylation.
Relative catalytic activity is expressed as percentage compared to the non-conjugated L-asparaginase defined as 100% and reported by each study. Modification degree refers to the average percentage of conjugated sites relative to the available amine groups per L-asparaginase tetramer. The results to generate this figure were extracted from references [16–22,34] and this work.
Fig 5.
Reusability of the 1kDa-PEG-conjugate.
Relative catalytic activity at temperatures ranging from 20°C to 80°C. The 1kDa-PEG-conjugate (in green), native L-asparaginase (in blue), and a commercial randomly-PEGylated L-asparaginase formulation (Millipore Sigma, USA) (in black). Relative catalytic activity was calculated by the absorption at 425 nm divided by the maximum signal for each sample and expressed as percentile (%). Reported values are average with error bars representing the 95% confidence interval. For the 1kDa-PEG-conjugate the same sample (semi-solid gel) was used throughout all the experiment.
Table 3.
Extracytoplasmic secretion of recombinant L-asparaginase constructs.
Table 4.
Catalytic activity of L-asparaginase C77-105S mutant.
Fig 6.
Computational analysis of the C77-105S mutation.
(A) Superposition of geometrically optimized structures of the natural disulfide bond and the C77-105S mutation. (B) Formation of a new hydrogen bond in the C77-105S mutant.
Fig 7.
Subunit mass of recombinant native and mutant A38C-T263C L-asparaginase.
The m/z peak for the native L-asparaginase subunit (in green) was observed at 34605 g/mol, while the m/z peak for the mutant A38C-T263C subunit (in black) was observed at 34634 g/mol.