Part:BBa_K3895010

kerBlER-15_dna_pET-28a(+)

Construction

Keratinase KerBIER15 (BBa_K3895003) is originally extracted from Bacillus licheniformis ER-15. It is a serine keratinase and most active in the pH range of 8-12 and at 60°C [1]. The keratinase was compared with the other 3 keratinases (BBa_K3895003; BBa_K3895004;BBa_K3895005) with the same construction method. 6x His-tags were added to both sides of the sequence for purification and constructed into PET-28a(+). The constitutive T7 promoter (BBa_I719005) derived from the T7 bacteriophage allows high expression of proteins.

Protein purification

Name of keratinase	IPTG concentration/mM	Temperature/°C	Time/h
KerBIER15	0.4196	37	16

Figure 1. PCR and gel electrophoresis of KerBIER15.

Figure 2. SDS-PAGE of purified protein.Protein molecular marker; Lane1. Empty PET-28a vector; Lane2. The crude enzyme (kerBlER15); Lane3. The crude enzyme (kerAvDZ50); Lane4. The purified enzyme (kerBlER15); Lane5. The purified enzyme (kerAvDZ50); Lane6. Empty PET-28a vector; Lane7. The crude enzyme (kerBlMKU3); Lane3. Crude enzyme (kerBteQ7); Lane8. The purified enzyme (kerBlMKU3); Lane5. The purified enzyme (kerBteQ7).

Protein Concentration

Figure 4. Protein concentrations of 400 times dilute of the 4 ultrafiltrated keratinases calculated based on BCA standard concentration curve.

Protocol - BCA test for protein concentration [2] Protein standard solution preparation: Add 0.8ml protein standard to 20mg BSA, fully dissolve the solid to get 25mg/ml protein standard sol (can be stored in -20°C) Dilute some 25mg/ml Protein standard to 0.5mg/ml (eg. take 25ul 25mg/ml protein standard sol, add 980ul diluent to get 0.5mg/ml Protein standard ) Use PBS as diluent (0.5mg/ml Protein standard sol can be stored in -20°C too)

BCA working solution preparation: Add 1 volume of BCAreagent B to every 50 volumes of BCAreagent A(1:50) to get BCA working sol, mix them together (stable in room temperature for 24hrs)

Protein concentration test: Add protein standard to the 96 pore plate (according to 0, 1, 2, 4, 8, 12, 16, 20ul), add the diluted protein standard to each pore until 20ul (20, 19, 18, 16, 12, 8, 4, 0ul). Add the 20ul sample( what we want to measure) to the 96pore plate well, record the sample volume (if the sample volume is less than 20ul, add diluted protein standard to it until 20ul ) Add 200ul BCA working sol to each pore, set it aside for 20-30min at 37°C/ 2hrs at room temp/ 30min at 60°C Measure A562 or absorbance at 540-595nm Calculate protein concentration using the standard curve and sample volume.

Activity Comparision

Figure 5. The activity of the keratinase on different substrates. An increase in 0.01 OD was considered as 1 unit of enzyme activity.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 606
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 411
  Illegal AgeI site found at 1149
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 351