Biotechnology Letters 26: 753–756, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
753
Chemical modification of L-asparaginase from Escherichia coli with a modified polyethyleneglycol under substrate protection conditions Jian-Feng Zhang, Ling-Yang Shi & Dong-Zhi Wei∗ State Key Laboratory of Bioreactor Engineering, Research Institute of Biochemistry, East China University of Science and Technology, Shanghai 200237, P.R. China ∗ Author for correspondence (Fax: +86-21-64250068; E-mail:
[email protected]) Received 7 January 2004; Revisions requested 4 February 2004; Revisions received 4 March 2004; Accepted 5 March 2004
Key words: L-asparaginase, chemical modification, polyethyleneglycol
Abstract L -Asparaginase was chemically modified with 2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-S-triazine (mPEG2 ) in the presence of L-asparagine. Optimal modification was performed under the condition that the molar ratio of mPEG2 /-NH2 is 10. The modified enzyme retained 33% of initial enzymatic activity with complete abolishment of immunogenicity. In vitro half-life increment from 4.6 h to 33 h has been obtained.
Introduction L -Asparaginase (E.C. 3.5.1.1) is a potential therapeutic agent for acute lymphocytic leukaemia, acute lymphoblastic leukaemia and chronic myelogenyous leukaemia. However, native asparaginase is associated with a high incidence of allergic reactions. Much effort has been devoted to developing methods to avoid such side-effects as well as to increasing its in vivo half-life. For example, L-asparaginase has been chemically modified by polyethyleneglycol (Matsushima et al. 1980, Soares et al. 2002), poly-(D,L-alanine) (Uren et al. 1982), dextran (Wileman et al. 1986), and polyvinyl-pyrrolidone (Qian et al. 1997). Chemical attachment of polyethyleneglycol (PEG) to therapeutic proteins bestows several benefits, such as enhanced plasma half-life, decreased toxicity, and increased drug stability and solubility (Roberts et al. 2002). To ensure mild modification conditions, there requires a suitable activated species. One good example is mPEG2 , generated from mono-methoxypolyethyleneglycol (mPEG) and cyanuric chloride (Matsushima et al. 1980). Modification of L-asparaginase with mPEG leading to low antigenicity but low enzymatic activity has previously been reported (Ashihara et al. 1978). Here we present a new method to attain zero immunogenicity with a high re-
sidual activity through modification of L-asparaginase with mPEG2 in the presence of L-asparagine.
Materials and methods General L -Asparaginase from E. coli was produced in our laboratory. Protein concentration was determined by the Bradford method. SDS-PAGE analysis was performed by the Laemmli procedure using 12% polyacrylamide gel and staining with silver nitrate. Monomethoxypolyethyleneglycol (MW5000), Sephadex G150, and Coomassie Brilliant Blue G-250 were from Sigma. Fluorescamine and cyanuric chloride were products of Acros Co., Belgium. Freud’s adjuvant was provided by Shanghai Bioengineering Center, Chinese Academy of Science.
Determination of L-asparaginase activity L -Asparaginase activity was assayed as follows (see Matsuyama et al. 1991). In brief, 0.25 ml 40 mM L -asparagine was added to 0.75 ml protein solution (6 mg ml−1 in 0.1 M potassium phosphate buffer, pH 7) and preheated at 37 ◦ C for 5 min. The reaction
754 Table 1. Effect of mPEG2 amount on the activity of the modified enzyme. mPEG2 /-NH2 a
2.5
5
7.5
10
12.5
15
20
30
Residual enzymatic activity (%)b
74
41
38
33
30
21
10
0
a The entry stands for the molar ratio of modifier molecules to free amino groups in native
enzymes (each native enzyme molecule contains 92 free amino groups). b Enzymatic activity assay was performed according to the method from the literature
(Matsuyama et al. 1991). Residual enzymatic activity is expressed as the percentage of modified protein enzymatic activity to native proteins. The enzymatic activity of native L-asparaginase was 350 IU/mgPro.
was terminated with 0.25 ml 15% (w/w) trichloroacetic acid followed by centrifugation (100 g, 5 min). Supernatant, 0.25 ml, was transferred into a new tube and treated with 0.5 ml Nessler’s reagent for 15 min at room temperature. The absorbancy of the sample was then measured at 500 nm. A working curve was obtained under identical conditions with ammonium sulfate as a standard.
Table 2. Affinity to antibody of native enzyme and modified enzymea .
Chemical modification of L-asparaginase with mPEG2
a Double diffusion immunization (Matsushima et al. 1980) was
mPEG2 was synthesized and purified according to the procedure (Matsushima et al. 1980). L-Asparaginase, 2 mg in 1 ml 0.1 M H3 BO3 /NaOH buffer (pH 10), was mixed with 20 µl 6 mg L-asparagine ml−1 in the same buffer. The mixture was incubated at 37 ◦ C for 0.5 h, and a stoichiometric amount of mPEG2 (in molar ratio of mPEG2 /-NH2 ) was introduced. The sample was incubated at 37 ◦ C for 1 h followed by adding 10 ml 0.1 M 0 ◦ C potassium phosphate buffer (pH 7) to terminate the reaction. The protein was then dialyzed overnight by 0.1 M H3 BO3 /Na2 B4 O7 buffer (pH 8.5) and purified afterwards on Sephadex G-150 column eluting with 0.1 M potassium phosphate buffer (pH 7).
mPEG2 /-NH2 b
0 2.5 5 7.5
Diluted multiples of the antiserum 21
22
23
24
25
26
++ ++ ++ ++
++ ++ ++ +
++ ++ + +
++ + + −
++ + − −
++ − − −
adopted. ‘++’ Stands for clear sediment, ‘+’ stands for sediment can be seen, ‘−’ stands for no sediment. b The entry stands for the molar ratio of mPEG molecules to free 2 amino groups in initial L-asparaginases. There is no reaction for the modified enzyme of molar ratio equal to or greater than 10 on any diluted multiples of the antiserum.
Determination of enzyme’s affinity to antiserum Enzyme’s affinity to antiserum was determined by double diffusion immunization (Matsushima et al. 1980). A mixture of 3% (w/v) agar with equal volume of 0.05 M barbital buffer (pH 7.4) was dripped into a clean plate, baked to dryness, cooled to room temperature, and then followed by perforating 7 cells of 3 mm diam. and 4 mm spacing interval with one on the center and the other six equidistantly distributed around the center cell. Ten µl enzyme sample was dripped into the center cell and the same amount of multiply diluted different antiserum was dripped into
Fig. 1. SDS-PAGE analysis of the native and modified L-asparaginase. Lanes 1, 3: 0.5 mg ml−1 modified proteins (mPEG2 /-NH2 = 10, molar ratio); lanes 2, 4: 0.1 mg ml−1 native proteins (standard); lanes 1, 2: 10 µl dosage; lanes 3, 4: 20 µl dosage. Bands at site B conform to the subunit mass of E. coli L-asparaginase. Bands at site A are the modified enzyme with an estimated molecular weight of 39 kDa.
each of the six cells. The plate then was incubated at 37 ◦ C for 24 h to determine antigenicity.
755 suggest that the attachment of mPEG2 to the amino groups on the protein surface has been successfully achieved. The elution profile of the modification mixture on Sephadex G-150 column is presented in Figure 2. The first peak corresponds to the modified L-asparaginase, whereas the second is the native protein. A good separation efficiency was achieved and both protein fractions exhibited hydrolysis activity on L-asparagine. Effect of mPEG2 amount on the enzymatic activity Fig. 2. Separation of native and modified enzyme using Sephadex G-150 (2.5 × 30 cm). Two mg L-asparaginase was modified with mPEG2 at the molar ratio of 10 (mPEG2 /-NH2 ), and then introduced to the column for separation after dialysis. 0.1 M potassium phosphate buffer (pH 7) was used as the eluant at a rate of 0.25 ml min−1 . The eluate was collected with absorbancy determination at 280 nm.
Modified L-asparaginase with different extent of mPEG2 attachment was obtained by changing the molar ratio of mPEG2 /-NH2 in the reaction. Results of enzymatic activity with different extents are summarized in Table 1. In general, the enzyme gradually loses activity upon modification. This tendency can be explained by conjugation of mPEG2 with free amino groups on the protein surface, which results in steric impediment that prevents asparagine from approaching the enzyme active site. As shown by Ashihara et al. (1978), it is imperative to have conjugation in the presence of L-asparagine to preserve higher residual activity. Variation of affinity to antiserum
Fig. 3. Half-life of native () and modified enzyme () in vitro. Enzyme in 0.1 M borate buffer (pH 8.5) was incubated at 37 ◦ C for the times shown and then assayed for activity.
Results and discussion SDS-PAGE analysis and chromatography separation L -Asparaginase was modified by monomethoxypolyethyleneglycol of 5000 Da (Ashihara et al. 1976). SDS-PAGE analysis of the modification protein is shown in Figure 1. More bands are found in the modified protein samples (lanes 1, 3) than in the native samples (lanes 2, 4). Site B gives the single band of the native protein, which matches well with the subunit mass of E. coli L-asparaginase (34 kDa). Bands at site A show the modified enzymes with an estimated molecular weight of 39 kDa. Such distinguishable bands
The enzyme’s affinity to antiserum by double diffusion immunization was summarized in Table 2. The binding ability of the modified enzyme to antiserum decreases gradually with the increased amount of mPEG2 . When the molar ratio of mPEG2 /-NH2 is equal to or greater than 10, no discernible sediment can be observed, suggesting that its immunogenicity has been completely abolished. Our result suggests that the optimal modification be performed under the condition that the molar ratio of mPEG2 /-NH2 is 10. Under such a condition, the modified enzyme retains 115 IU/mgPro activity, which is 33% of the native 350 IU/mgPro. Half-life of the native and modified enzyme A longer half-life for L-asparaginase is desirable because the plasma can thus maintain a higher concentration for clinical effectiveness. It is well known that conjugation of L-asparaginase with mPEG2 can improve thermostability and stability due to the beneficial effects from the mPEG chains. It is reasonable to deduce that such beneficial effects may also lead to increment of in vitro half-life. Our experimental
756 results are shown in Figure 3. The half-life of native and mPEG2 -conjugated enzyme is estimated to be 4.6 h and 33 h, indicating that our modification has achieved 6-fold increment of half–life for native L -asparaginase.
Acknowledgement This work was supported by the Key Disciplinary Foundation of Shanghai, P.R. China.
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