In Silico Methods for Mutational Analysis of Ocriplasmin Variants
The undesirable autolytic and proteolytic activities of ocriplasmin can be reduced with site-directed mutagenesis leading to an enzyme with improved catalytic activities and half-life. Three ocriplasmin variants were designed for improving biological/physicochemical characteristics. Using Swiss Model (42) and I-TASSER (43) web servers, 3D Structural Modelling of ocriplasmin was done. After that, the position of A59T and K156E mutations was studied by SPDBV (44) and PyMOL (45) software. Subsequently, the structural analyses of wild type and three mutants variants were performed using a protein interaction calculator (PIC) server available at crick.mbu.iisc.ernet.in/PIC (46). A molecular docking simulation was performed using the freely available package Auto Dock 4.2.6 and Auto Dock Tools 1.5.6 software to study the interactions between substrate S-2403 (L-pyroglutamyl-L-phenylalanyl-Llysine-p-nitroaniline hydrochloride, Chromogenix, Milano, Italy, cat. 822254-39 as a chromogenic substrate for plasmin, ocriplasmin, and streptokinase-activated plasminogen) and ocriplasmin variants and the binding capability was evaluated by calculating the free binding energy. The conformational features of protein-substrate complexes for all the variants were evaluated in a 100-ns Molecular Dynamics Simulation using the GROMACS 2016 package (R. Baghban, unpublished work).
Construction and Cloning of the Ocriplasmin Expression Vector
The ocriplasmin coding sequence (DB08888 (DB05028)) was codon-optimized and synthesized (Genscript, U.S.A) for expression in P. pastoris. For subcloning into the expression vector, the ocriplasmin gene was amplified by polymerase chain reaction with the primers forward: 5′-CTATTGCCAGATTGCTGC-3′ and reverse: 5′-GCGTGAATGTAAGCGTGAC-3′ using Taq DNA polymerase (Vivantis, Gyonggi, South Korea). PCR protocol was carried out with initial denaturation at 94 °C for 4 min followed by, denaturation at 94 °C for 1 min, annealing at 55 °C for 45 Sec, extension at 72 °C for 45 Sec, and a final extension at 72 °C for 5 min. KpnI and XhoI (TaKaRa Biotech, Japan) restriction enzymes were used to digest the purified PCR product and then ligated into the pPinkα-HC vector (Thermo Fisher Scientific, U.S.A) under the control of the alcohol oxidase I (AOX1) promoter. Afterward, the pPinkα-HC-ocriplasmin ligation product was transformed into the E. coli DH5-alpha (Thermo Fisher Scientific, U.S.A). To select the clones, the transformants were plated onto Luria-Bertani (LB) agar containing 100 mg/ml of ampicillin (Sigma-Aldrich, U.S.A). The recombinant constructs extracted from the ampicillin-resistant clones were confirmed with colony PCR and restriction digestion.
Mutant Construction Using Site-directed Mutagenesis
The optimized coding sequence of the ocriplasmin gene was employed as a template for the amplification. Glutamic acid was substituted for lysine 156 (K156E) in SOEing PCR to construct the mutant variant and reduce autolytic activities. Amplification was first performed on a 481 bp DNA fragment, containing the 5′ end of the ocriplasmin gene and upstream sequences (with α factor-F1 and OCR-R2 primers), and a 330 bp fragment, including the 3′ end of the gene and downstream sequences (with OCR-F2 and CAC1-R1 primers) following the PCR protocol of the previous section. To generate mutated fragments containing the desired point mutations, in the second round, amplified fragments were joined by overlap extension PCR method (47) according to the following temperature profile: initial denaturation at 94 °C for 5 min, annealing at 57 °C for 2 min, and extension at of 72 °C for 2 min. The reaction was followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 57 °C for 45 sec, extension at 72 °C for 1 min, and a final extension at 72 °C for 5 min.
Alanine 59 changed to threonine (A59T) to construct the mutant fragment and reduce proteolytic activities. Amplification was performed on a 190 bp DNA fragment, including the 5′ end of the ocriplasmin gene and upstream sequences (with α factor-F1 and OCR-R3 primers) and a 617 bp fragment, including the 3′ end of the ocriplasmin gene and downstream sequences (with OCR-F3 and CAC1-R1 primers). Splicing by overlap extension was performed in the second round of PCR to join these fragments together (47) and generate a PCR fragment including the desired point mutations.
To produce the mutant fragment containing both the mutations, A59T and K156E, the fragment with A59T mutation was used as a template for adding K156E change as per the same steps described. These mutated PCR fragments were cloned into XhoI and KpnI restriction sites of the pPinkα-HC vector and transferred into the E. coli DH5-alpha. PCR and plasmid digestion confirmed the cloning and sequencing confirmed the mutations.
Transformation and Selection in P. pastoris
The recombinant pPinkα-HC-ocriplasmin vectors extracted and linearized with SpeI (TaKaRa Biotech, Japan). P. pastoris strain 4 competent cells (Thermo Fisher Scientific, U.S.A) were prepared and transformed with 5-10 μg linearized plasmids by electroporation for the wild type and three mutant variants of ocriplasmin. The transformations were recovered at 30 °C in yeast extract peptone dextrose with sorbitol (YPDS) for 2-12 hours. The cell mixture spread on minimal dextrose (MD) plates were incubated at 30 °C for 3-10 days to obtain distinct colonies. Colony PCR was performed using specific primers to confirm the clones that integrated heterologous expression cassettes.
Small Scale Expression and Optimization in P. pastoris
For the small-scale expression, the wild-type and mutant colonies inoculated from the fresh transformation plates in 10 ml BMGY medium (buffered glycerol-complex medium yeast) containing 1 % (w/v) yeast extract, 2 % (w/v) peptone, 100 mM potassium phosphate, pH 6.0, 1.34 % (w/v) yeast nitrogen base (YNB), 0.0004 % (w/v) biotin, and 1 % (w/v) glycerol in a shaking incubator for 1-2 days at 24-30 °C until an optical density (OD600) of 4-6 was obtained. After transferring the cells into 50-ml of fresh BMGY medium, they were incubated for one day until obtaining an OD600 of 5-6. To induce the ocriplasmin expression, the cells were transferred to BMMY medium (buffered methanol-complex medium yeast with the same composition of BMGY except for glycerol, which is replaced with methanol). The induced cultures underwent a four-day incubation at 150 rpm and 30 °C and were fed, every 24 hours, with methanol at a final concentration of 0.5 %. The supernatant was collected at 24, 48, 72, and 96 hours for analyzing the protein expression.
The culture conditions under which the ocriplasmin expression was optimized in the adenine-deficient P. pastoris strain included cell density=one and two-fold, pH=3-7, temperature=20, 25, and 30 °C, concentrations of glycerol=5-10 %, methanol=0.25-8% and ammonium sulfate=5-20% and induction time=1-4 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were performed to analyze the samples of the supernatants.
Production of Anti-Plasminogen Polyclonal Antibody in Rabbit
Anti-plasmin antibodies can help detect ocriplasmin as a truncated version of recombinant human plasmin. A male New Zealand white rabbit was immunized with four intradermal injections on days 1, 14, 23, and 31. The first and the following three injections comprised a mixture of 150 μg of plasminogen (Merck, Germany) in 0.3 ml of saline respectively emulsified in 0.3 ml of Freund's Complete Adjuvant and Freund's Incomplete Adjuvant (Sigma-Aldrich, Germany ). The blood samples collected from the ears of the rabbit before every immunization and kept for two hours at 4 °C for coagulation. The serum separated from the coagulated blood was kept at -20 °C. Western blotting and enzyme-linked immunosorbent assay (ELISA) were performed to quantitatively and qualitatively evaluate the produced antibody.
Specificity Determination
The specificity of interactions between ocriplasmin expressed in P. pastoris and anti-plasminogen antibody produced in rabbits was determined using ELISA. The wells were coated overnight with 10 μg/ml of recombinant ocriplasmin in carbonate-bicarbonate buffer with pH 7.2 at 4 °C. The plates were rinsed three times using phosphate-buffered saline (PBS) with 0.05 % Tween 20. 100 µl of different dilutions (1/400, 1/800, 1/1600, 1/3200, 1/6400, 1/12800, 1/25600) of rabbit serum containing the anti-plasminogen antibody was added to the wells. After one-hour incubation, the plates were rinsed and incubated for one hour using 1: 5,000 dilutions of the HRP-conjugated anti-rabbit antibody (Sigma-Aldrich, Germany). The TMB (Sigma-Aldrich, U.S.A) substrate was added after washing, and the reaction was stopped 20 min later using a 1 N H2SO4. The absorption was ultimately read at 450 nm.
Immunoblotting
The recombinant ocriplasmin secreted from P. pastoris underwent immunoblotting by separating cell lysate proteins through SDS-PAGE and then transferring them to a polyvinylidene difluoride membrane (Thermo Fisher Scientific, U.S.A). A solution of 3 % (w/v) bovine serum albumin in TBS (tris-buffered saline) with 0.1 % Tween 20 blocked the membrane. Ocriplasmin was detected using an anti-plasminogen antibody produced in rabbit and HRP-conjugated goat anti-rabbit IgG as a secondary antibody (Sigma-Aldrich, U.S.A). Enhanced chemiluminescence (ECL) (Thermo Fisher Scientific, U.S.A) was then used to detect proteins.
Activation of Ocriplasmin Variants
Since the ocriplasmin and their variants expressed in Pichia were in the inactive zymogen forms, it was necessary to be activated by the use of urokinase plasminogen activator (Sigma-Aldrich, U.S.A) to convert the purified ocriplasminogen variants into the corresponding active forms. We used different enzyme concentrations and incubation times to achieve the optimal time and concentration of urokinase enzyme for the activation of ocriplasmin. Solutions of the ocriplasminogen variants with concentrations of 5-20 mM were incubated at 37 °C for 10, 30 and, 60 minute, in the presence of the urokinase at an ocriplasminogen to urokinase ratio of 100/2 and 100/5. After completing the phase of activation, SDS-PAGE was performed to evaluate the conversion of ocriplasminogen to the active form.
Measuring Hydrolytic Activities
The hydrolytic activities of active ocriplasmin species extracted were monitored against S-2403™ (Chromogenix, Milano, Italy) as a substrate explained by Aerts et al. in 2012 (28). The initial release rate of p-nitroaniline was monitored at 405 nm using Chromogenix S-2403™ to measure the hydrolytic activity of ocriplasmin. 1-10 nM ocriplasmin and 0.3 mM S-2403 substrate were used. Finally, the measured activities of wild type were compared with the hydrolytic activities determined for the mutant variants.
Characterizing the Kinetic Parameters; the Michaelis Constant (Km), the Catalytic Rate (Kcat), and Vmax
Initial hydrolysis rates were measured at different concentrations of S-2403 as the substrate to obtain the kinetic parameters of ocriplasmin variants. The mutant and wild-type enzymes were aliquoted in a duplicate manner to volumes of 75 μl to obtain a fixed concentration and the substrate was added at concentrations of 2.4, 1.2, 0.6, 0.3, 0.15, and 0.075 mM. After measuring the absorbance at 405 nm every 5 minutes, a standard curve was employed to calculate the micromoles of the released product per minute. Linear regression was used to calculate Kcat, Km, and Vmax based on equation (1), in which [S] represents the concentration of S-2403 and [OCR] that of active ocriplasmin. Moreover, Kcat and Km could be calculated by analyzing total hydrolysis curves derived at [S] and Vmax using equation (2). The data were fitted to the Michaelis–Menten equation using GraphPad Prism software (version 8.0.2). The measurements were performed at 37 °C and in a mixture of 38 mM NaCl, 50 mM Tris-HCl, and 0.01 % Tween 80 with pH=7.4.