DOI: 10.4103/1735-5362.383709 ISSN:

A novel approach to enhance the performance of kallikrein 6 enzyme using Pichia pastoris GS115 as a host

Fatemeh Mahmoodi, Hamid Bakherad, Navid Mogharrab, Mohammad Rabbani
  • General Pharmacology, Toxicology and Pharmaceutics

Background and purpose:

Enzyme engineering is the process of raising enzyme efficiency and activity by altering amino acid sequences. Kallikrein 6 (KLK6) enzyme is a secreted serine protease involved in a variety of physiological and pathological activities. The increased expression of KLK6 plays a key role in various diseases. Instability and spontaneous activation and deactivation are major challenges in the study of this enzyme. This study aimed to create a stable pro-KLK6 enzyme by enzyme engineering, designing a specific cleavage site for enterokinase, and using Pichia pastoris GS115 as a host cell. Then, recombinant pro-KLK6 was used to introduce a novel inhibitor for it.

Experimental approach:

An engineered pro-KLK6 gene was cloned into the pPICZα A expression vector. Then, it was expressed in P. pastoris GS115 and purified by Ni-NTA chromatography. An inactive engineered pro-KLK6 gene was cleaved by enterokinase and converted to an active KLK6. The KLK6 enzyme activity and its kinetic parameters were measured using N-benzoyl-L-arginine ethyl ester (BAEE) substrates.


The secretory form of the pro-KLK6 was expressed at about 11 mg/L in P. pastoris (GS115). Before activation with enterokinase, pro-KLK6 was inactive and did not activate spontaneously. The kinetic parameters, including Km and Vmax, were estimated at 113.59 μM and 0.432 μM/s, respectively.

Conclusion and implications:

A stable pro-KLK6 enzyme was produced using P. pastoris (GS115) as the host cell and a specific cleavage site for enterokinase. Additionally, this study assessed the kinetic parameters of the KLK6 enzyme using the BAEE substrate for the first time.

More from our Archive