Directed Evolution of an Antitumor Drug (Arginine Deiminase PpADI) for Increased Activity at Physiological pH

Abstract

Arginine deiminase (ADI; EC 3.5.3.6) has been studied as a potential antitumor drug for the treatment of arginine-auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. Studies with human lymphatic leukemia cell lines confirmed that ADI is an antiangiogenic agent for treating leukemia. The main limitation of ADI from Pseudomonas plecoglossicida (PpADI) lies in its pH-dependent activity profile, its pH optimum is at 6.5. A pH shift from 6.5 to 7.5 results in an approximately 80 % drop in activity. (The pH of human plasma is 7.35 to 7.45.) In order to shift the PpADI pH optimum, a directed-evolution protocol based on an adapted citrulline-screening protocol in microtiter-plate format was developed and validated. A proof of concept for ADI engineering resulted in a pH optimum of pH 7.0 and increased resistance under physiological and slightly alkaline conditions. At pH 7.4, variant M2 (K5T/D44E/H404R) is four times faster than the wild-type PpADI and retains approximately 50 % of its activity relative to its pH optimum, compared to approximately 10 % in the case of the wild-type PpADI.

Fluorescent Assay for Directed Evolution of Perhydrolases

“…Screening systems with a standard deviation lower than 14% are routinely and successfully used in directed evolution experiments. 33 Subtilisin Carlsberg is furthermore the first protease engineered through a directed evolution experiment toward increased perhydrolytic activity, which offers novel opportunities for bleaching applications. The protease variant, T58A/G165L/L216W, identified in the current work showed a 9.4-fold increased specificity constant of perhydrolytic activity compared with the wild type. )…”

A first continuous 4-aminoantipyrine (4-AAP)-based screening system for directed esterase evolution

“…For the first time, a continuous monitoring of esterase activity for phenol is reported which can likely be adapted to complex aromatic substrates with nonsubstituted p-positions (Wong et al 2005). The general applicability of the cBLE-4AAP detection system was evaluated by determining key performance parameters for a robust and reliable high throughput screening system: a low standard deviation (<14 % at 55°C (Zhu et al 2010) and 5 % at RT), minimal background, high sensitivity (15 μM), broad linear detection range (15-250 μM) and broad pH profile (7.4-10.4). In order to validate the cBLE-4AAP detection system, a first proof of concept was performed through a single round of directed evolution in which the residual activity of pNBEBL was improved after a thermal denaturation.…”

“…2). Screening systems with standard deviation lower than 14 % are routinely employed in successful directed evolution campaign (Zhu et al 2010). Interferences of each reaction component (i.e., pNBEBL, benzoic acid, and phenol) within the cBLE-4AAP detection system and the influence of pH are shown in Fig.…”

“…A random pNBEBL mutant library was generated and screened for improved thermal resistance using phenyl benzoate as substrate. epPCR library (Cadwell and Joyce 1992) (step 1) containing 0.04 mM MnCl 2 resulted in ∼50 % inactive clones, which represents a mutational load of one to two amino acid substitutions per gene (Zhu et al 2010). Prescreening was performed on tributyrin agar plates to select active pNBEBL variants for efficient screening in MTPs (step 2).…”

Directed evolution 2.0: improving and deciphering enzyme properties

“…71 Zhu and Cheng et al 11,65,66 used the 'KnowVolution' strategy to improve PpADI's specific activity at low arginine concentrations (0.1 mM under physiological conditions in blood) and to gain molecular understandings to shift the pH-optimum and to decrease the S 0.5 ('K m ') value of PpADI. In phase I, three iterative rounds of epPCR and one round of SeSaM were performed and in total 9300 clones were screened.…”