Part:BBa_K3195003

His-tagged Cathepsin B

This Cathepsin B from Branchiostoma belcheri tsingtauense is originally a kind of hydrolase in lysosome.In our project,it has been used to biodegrade microcystin,a kind of biotoxin synthesized by cyanobacteria.

Usage and Biology

Our BioBrick encodes a kind of cathepsin from Branchiostoma diverticulum epithelial cells. Cathepsin is a class of proteins found in the cells of various animal tissues, especially lysosome parts. Based on the results of bioinformatics analysis, the protein we found out belongs to cathepsin B. In Branchiostoma diverticulum epithelial cells, we speculate this kind of protein plays an important role in degrading cyanobacteria and microcystin. In fact, according to our experimental result, Cathepsin B protein has high efficiency in degradation of microcystin LR. So we construct a composite part so that other teams can easily use it. This BioBrick can be implemented in any host expression system by cloning it into an appropriate vector.

Figure 1..The abscissa represent 28 enzymes in our project and the unit of ordinate value is mg/L.The sample of MC-LR/RR degraded respectively by 28 enzymes was measured by HPLC-MS-MS and the results are as shown. The ordinate value represents the remained microcystin in the degraded sample.The sample 2 was degraded by cathepsin B. It shows that Cathepsin B is the most effective enzyme among the 28 proteins in degradation of MC-LR.

Characterization

Expression

The Biobrick was cloned in pET28b expression vector. After confirming the cloning by sequencing, the plasmid was transformed into E.coli DH5α. The transformation was confirmed by colony PCR.

Cells (E. coli BL21 (DE3)) were cultured with the induction of IPTG under optimal expression condition. Ultrasound broke cells to separate proteins. A special sequence, His-Tag, was added to the end of the target protein. His-Tag can bind to metal Ni2+ ions, which is beneficial to the purification of target protein. The protein added with His-Tag can be purified by Ni2+ affinity chromatography column under non-denaturing conditions. SDS-PAGE was used to detect the expression of proteins after purification. The result was below.

Figure 2Final sample QC of denatured condition. Coomassie blue staining.Reducing-PAGE analysis. 2µg of sample loaded.1. Before dialysis 2. After dialysis

Activity assay

The BioBrick was characterized by measuring cathepsin B activity using Cathepsin B Activity Assay Kit. BioVision's Cathepsin B Activity Assay Kit is a fluorescence-based detection technique. Using AFC (7-amino-4-trifluoromethyl coumarin) labeled cathepsin-B priority substrate sequence RR. Cell solutes or other samples containing Cathepsin-B can digest RR-AFC and release free AFC. Free AFC can be easily quantified by fluorometer or fluorescence microtitrator plate.

We determined Km and Vmax for our cathepsin B by performing non-linear regression using Michaelis-Menten model as below. The two parameters were: Vmax=3749.56222 ± 87.82359 RFU/h Km=0.22768 ± 0.02604 mol/L

Figure 2The non-linear fitting of Michaelis-Menten model

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 369
  Illegal XhoI site found at 958
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Unknown
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 463
  
• 1000
  COMPATIBLE WITH RFC[1000]