Part:BBa_K1378025

PelB-MlrA-Terminator

Introduction

MlrA coding sequence fused with PelB secretion signal peptide and terminator.

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]

Figure 1. First step of biodegradation of MC-LR. MlrA mediates breaking peptide bond between Adda and Arg, which leads to significant decrease of toxicity.^[1]

MlrA exhibits high degradation activity in lysis culture. Its activity in living cells, however, has no difference with control group. It indicates that our bacteria are unable to deal with MC immediately until they commit suicide. Thus, secretion system PelB is introduced. The PelB is linked to N-terminal of MlrA and the fusion protein is inserted into expression vector. We hope this measure would improve degradation effect largely in whole cell level.

Figure 2. Secretion sequence of MlrA. The fusion protein includes Type II secretion peptide pelB and MlrA. The construction as a whole is expressed in pET-21a(+) plasmid.

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 54
  Illegal AgeI site found at 310
  Illegal AgeI site found at 439
  
• 1000
  COMPATIBLE WITH RFC[1000]