Fig 1.
Purification of recombinant lumbrokinase.
(A) periplasmic extraction (B) cytoplasmic extraction.
Table 1.
Characteristics of Collected E. fetida.
Fig 2.
Map showing the sample collection site with coordinate generated by AutoCAD 2006.
Fig 3.
Morphological characteristics of locally isolated E. fetida.
(A) Morphology of the collected E. fetida (B) Dissected E. fetida showing internal visceral organ.
Fig 4.
1% agarose gel electrophoresis.
(A) Lane 2 represents total RNA extracted from earthworm. (B) PCR amplification product with LKF, LKR primers, Lane M; 1Kb DNA ladder, Lane 1: amplified PCR product.
Fig 5.
Lumbrokinase nucleotide (upper line) obtained from sequencing and deduced amino acid (lower line) sequences.
(GenBank: OP820958; Protein ID: WGV41589).
Fig 6.
Structural characteristics of lumbrokinase.
(A) The 2D structure prediction lumbrokinase (C represents the coils, H represents the helix and S represents the β-pleated sheets); (B) 3D model of lumbrokinase predicted by I-TASSER (C) Superimposed structure of lumbrokinase and its structural analogue1YM0A.
Fig 7.
Heterologous expression vector.
(A) recombinant pET22b-lumbrokinase (B) recombinant pET28-SUMO –lumbrokinase.
Fig 8.
Agarose gel electrophoresis of restriction analysis of recombinant vectors.
(A) pET22b (+) harboring lumbrokinase restricted with EcoR1 and HindIII; (B) pET28a SUMO harboring lumbrokinase restricted with EcoR1 and XhoI. Lane M: 1 kb DNA ladder; Lane 1: Amplified PCR product.
Fig 9.
SDS-PAGE analysis of total cell proteins from E. coli BL21 (DE3).
(A) harbouring pET22b (+)-lumbrokinase: Lane 1, uninduced sample; Lanes 2-5, samples induced with 1 mM IPTG at 28ºC for 2, 4, 8, and 10 hours, respectively; M, protein molecular weight marker. (B) harbouring pET28a (+) SUMO-lumbrokinase: Lane 1, uninduced sample; Lanes 2-5, samples induced with 1 mM IPTG at 37ºC for 2, 4, 6, and 8 hours, respectively; M, protein molecular weight marker.
Table 2.
Comparison of activities of lumbrokinase recovered from periplasmic space (expressed in pET-22b) and cytoplasmic space (pET28-a SUMO) from 1liter culture.
Fig 10.
SDS-PAGE of lumbrokinase at different stages of purification.
(A) expressed with pelB signal: CP, cytoplasmic protein; Un, uninduced sample; P, Periplasmic; (B) expression with SUOMO tag: In, induced sample; Un, un induced sample, H, His taq purified; U, final product after Ulp protease treatment; M, Protein marker.
Fig 11.
Fibrin plate assay a: Positive control (standard lumbrokinase), b: purified cytoplasmic recombinant lumbrokinase pET-28a (+) SUMO, c: Purified periplasmic recombinant lumbrokinase pET-22b (+), d: Negative control (1 × PBS buffer).
Fig 12.
Dose-time dependent blood clot lysis: recombinant lumbrokinase and standard lumbrokinase.
1X PBS Buffer negative control (2, 4, and 6 hours of incubation). (A) 0.5 mg/ml standard and recombinant lumbrokinase, tubes 1, 2, and 3 after 2, 4, and 6 hours of incubation, respectively. (B) 2.0 mg/ml standard and recombinant lumbrokinase, tubes 1, 2, and 3 after 2, 4, and 6 hours of incubation respectively.
Fig 13.
Percentage clot lysis activity: Dose-time dependent blood clot lysis of lumbrokinase.
Fig 14.
Graph represents the EC50 of recombinant lumbrokinase calculated by AAT Bioquest EC50 calculator.