Difference between revisions of "Part:BBa K1378001"

Revision as of 23:36, 17 October 2018

MlrA

Introduction

MlrA is a 28kDa protease found in Sphingomonas sp. which can cleavage microcystins(MCs).

MlrA is one part of the gene cluster responsible for the ability of MC degradation. The cluster includes four ORFs, mlrA, mlrB, mlrC and mlrD, which can hydrolyze MCs and facilitate absorption of the products as carbon source. MlrA is sometimes referred as a metalprotease by inhibitor studies.

MlrA can cleavage the Adda-Arg bond and causes ring opening.(Fig. 1) The first-step linearized product shows much weaker hepatoxin compared with MCs. In the experiment of mouse bioassay, up to 250 mg/kg of linearized MC-LR shows no toxicity to mouse, much higher than 50% lethal dose 50mg/kg of cyclic MC-LR. Furthermore, the linearization also raise the median inhibition concentration to 95nM, around 160 times higher than original 0.6nM. ^[1]

Characterization

1. Constructing method for analysis of MC concentration

p-Nitrophenyl phosphate (pNPP) is a widely used non-specific substrate to test protein phosphatase activity and it can be hydrolyzed to p-Nitrophenyl(pNP) with characteristic absorption at 405nm. The measurement of PP1 activity is based on the accumulation of pNP. Considering the microcystin (MC) is the inhibitor of PP1 and MlrA can disrupt MC’s structure to disrupt its inhibitory effect, the MlrA activity can be detected by quantification of absorption at 405nm (Fig. 2). So the concentration of MCs after degradation can be finally measured by absorption spectrophotometry method with all the calibration curves for all the interactions above.

Firstly a calibration curve of PP1 activity was generated. The concentration of substrate pNP is sufficient overall so the PP1 enzyme is saturated and proportion to the accumulation rate of product pNPP. We could select a proper working concentration of PP1 in the range of nearly linear relationship between PP1 and change rate of 405nm absorption.

Based on the premise of linear relationship between product and absorbance, we choose 0.05unit/ul as the working concentration of PP1 and then test the inhibition efficiency of MC-LR. As a result, PP1 activity decreases after the addition of MC-LR and there is a positive correlation between the reduction of absorbance and concentration of MC-LR.

2. Verifying the degradation effect of MlrA

To test the degradation efficiency of MlrA expressed by E. coli, MlrA expression plasmid has been constructed and transformed into E. coli strain BL21(DE3) (Fig. 5a). After induction, the bacteria are lysed by lysozyme and incubated with MC solution. Judged by PP1 activity treated by the mixture, the activity in experiment group expressing MlrA is much higher than strain carrying blank vectors, suggesting that MC-LR is degraded (Fig. 5b). Therefore, it could be concluded that MlrA function well in E. coli expression system.

References

[1] Gehringer, M. M., Milne, P., Lucietto, F., & Downing, T. G. (2005). Comparison of the structure of key variants of microcystin to vasopressin. Environmental toxicology and pharmacology, 19(2), 297-303.

Contribution

Aalto-Helsinki 2016 team has contributed to this part by improving its sequence and characterization. The part number is BBa_K1907002.

We improved parts mlrA gene BBa_K1378001 by modifying its sequence, adding 6x His tag, as well as making it a segment of composite parts. Its part number is BBa_K2888010.

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