Protease variants
A protease variant, protease technology, applied in the field of protease variants, can solve problems such as incorrect three-dimensional structure
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 2
[0476] Method for producing protease variants
[0477] The invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell which has been transformed with a DNA sequence encoding the enzyme is cultured under conditions permitting production of the enzyme, and the resulting enzyme is recovered from the culture.
[0478] Heterologous recombinant production of the enzyme of the invention is possible when an expression vector comprising a DNA sequence encoding the enzyme is transformed into a heterologous host cell. It is thus possible to prepare highly purified RP-II protease compositions characterized by the absence of xenogeneic impurities.
[0479] The medium used for culturing transformed host cells may be any conventional medium suitable for culturing the host cells. The expressed RP-II protease is routinely secreted into the culture medium and can be recovered therefrom by known methods including separation of the cells ...
Embodiment 1
[0513] Modeling of RP-II protease from the 3D structure of BLC
[0514] The overall homology of Bacillus licheniformis protease BLC with other RP-II proteases is high. Similarities between different RP-II proteases are provided in Table 1. use figure 2 The JA96 protease can be modeled using an appropriate modeling tool like Accellrys homology software, or Modeller (also from Accellrys), or other software like Nest. These programs provide results as an initial rough model, with certain optimizations in the Modeller and Nest programs.
[0515] The initial rough model ensures close structural homology between the JA96 protease model and the 3D structure of BLC, as there are no overlapping side chains in the model structure. To optimize structure, in silico proteins can be submerged in a water box and minimize energy, using for example Accelrys' CHARMm TM software for further molecular dynamics simulations. By adding a box volume of 75*75*75 in the Insight II program (purcha...
Embodiment 3
[0525] Purification of enzymes and variants
[0526] This procedure involves the purification of a 2 liter scale fermentation producing the protease RP-II of the invention in Bacillus host cells.
[0527] Approximately 1.6 liters of fermentation broth was centrifuged at 5000 rpm for 35 minutes in a 1 liter beaker. The supernatant was adjusted to pH 7 using 10% acetic acid and filtered through a Seitz Supra S100 filter plate.
[0528] At room temperature, the filtrate was applied to a 100 ml bacitracin affinity column equilibrated with 0.01M dimethylglutaric acid, 0.1M boric acid and 0.002M calcium chloride (buffer A), and the buffer was adjusted to pH 7. The column was washed with buffer A to remove unbound protein, and the protease was eluted from the bacitracin column using buffer A supplemented with 25% 2-propanol and 1M sodium chloride.
[0529] Fractions with protease activity eluted from the bacitracin purification step were pooled and applied to a 750 ml Sephadex G25...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More