Development of a cell-free protein synthesis protocol to rapidly screen L-asparaginase proteoforms by enzymatic activity

Abstract BACKGROUND Cell-free protein synthesis (CFPS) technology has emerged as a powerful tool for a variety of biotechnological applications, including the expression of different classes of biopharmaceutical products. L-Asparaginase (E.C. Number: 3.5.1.1, L-asparagine amidohydrolase) (L-ASNase) is an important biopharmaceutical used to treat leukemia, but expression of multiple proteoforms in CFPS systems and rapid characterization using standard colorimetric methods has not yet been fully exploited. Herein, recombinant expression and characterization of an L-ASNase from Erwinia chrysanthemi (Erwinase) using a new CFPS protocol is reported. RESULTS Expression and quantification of the enzymatic activity of a soluble his-tagged L-ASNase directly from a CFPS reaction was successfully achieved. Purification of the protein was not required in order to assess its biological activity. Activity of L-ASNase was significantly higher than the control reaction (7.07 ± 0.68 U mL–1 vs. 1.83 ± 0.14 U mL–1, respectively). Expression of a mutant Erwinase proteoform – V293M – was also achieved and it presented a similar enzymatic activity. No significant loss in L-ASNase enzymatic activity was noticed after removal of cyclic AMP, spermidine, transfer RNA, T7 RNA polymerase and, especially, ammonium acetate (a common interference in ASNase enzymatic assays) from the CFPS reaction. CONCLUSION The protocol developed in this work will facilitate the screening of novel clinically-relevant L-ASNase proteoforms. © 2021 Society of Chemical Industry (SCI).

Influence of lysosomal protease sensitivity in the immunogenicity of the antitumor biopharmaceutical asparaginase.

L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.

Heterologous expression and purification of active L-asparaginase I of Saccharomyces cerevisiae in Escherichia coli host.

l-asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc_ASNaseI and Sc_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc_ASNaseI (His)6 -tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract. Affinity chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using Ni2+ -charged, HiTrap Immobilized Metal ion Affinity Chromatography (IMAC) FF in order to purify active Sc_ASNaseI recombinant protein. The results suggest that the strategy for the expression and purification of this potential new biopharmaceutical protein with lower side effects was efficient since high amounts of soluble Sc_ASNaseI with high specific activity (110.1 ± 0.3 IU mg-1 ) were obtained. In addition, the use of FPLC-IMAC proved to be an efficient tool in the purification of this enzyme, since a good recovery (40.50 ± 0.01%) was achieved with a purification factor of 17-fold. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:416-424, 2017.

The promoter of filamentation (POF1) protein from Saccharomyces cerevisiae is an ATPase involved in the protein quality control process.

BACKGROUND: The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene. RESULTS: Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo. CONCLUSIONS: Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

The promoter of filamentation (POF1) protein from Saccharomyces cerevisiae is an ATPase involved in the protein quality control process

The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recentlybeen described as a cell component involved in yeast filamentous growth. The objective of this work is tounderstand the molecular and biological function of this gene.Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of theSaccharomyces cerevisiae protein quality control pathway. According to the results, Äpof1 cells showed increasedsensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such asdithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Äpct1, a strain thatlacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however,that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalyticefficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins(ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction betweenPof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.