Research Article: Efficient production of secretory Streptomyces clavuligerus β-lactamase inhibitory protein (BLIP) in Pichia pastoris

Date Published: April 20, 2018

Publisher: Springer Berlin Heidelberg

Author(s): Kin-Ho Law, Man-Wah Tsang, Yuk-Ki Wong, Ming-San Tsang, Pui-Yee Lau, Kwok-Yin Wong, Kwok-Ping Ho, Yun-Chung Leung.


β-Lactamase inhibitory protein (BLIP), a low molecular weight protein from Streptomyces clavuligerus, has a wide range of potential applications in the fields of biotechnology and pharmaceutical industry because of its tight interaction with and potent inhibition on clinically important class A β-lactamases. To meet the demands for considerable amount of highly pure BLIP, this study aimed at developing an efficient expression system in eukaryotic Pichia pastoris (a methylotrophic yeast) for production of BLIP. With methanol induction, recombinant BLIP was overexpressed in P. pastoris X-33 and secreted into the culture medium. A high yield of ~ 300 mg/L culture secretory BLIP recovered from the culture supernatant without purification was found to be > 90% purity. The recombinant BLIP was fully active and showed an inhibition constant (Ki) for TEM-1 β-lactamase (0.55 ± 0.07 nM) comparable to that of the native S. clavuligerus-expressed BLIP (0.5 nM). Yeast-produced BLIP in combination with ampicillin effectively inhibited the growth of β-lactamase-producing Gram-positive Bacillus. Our approach of expressing secretory BLIP in P. pastoris gave 71- to 1200-fold more BLIP with high purity than the other conventional methods, allowing efficient production of large amount of highly pure BLIP, which merits fundamental science studies, drug development and biotechnological applications.

Partial Text

β-Lactamase inhibitory protein (BLIP) is a low molecular weight protein (~ 17.5 kDa) naturally secreted by gram-positive bacterium Streptomyces clavuligerus (Doran et al. 1990). As its name suggests, BLIP can inhibit β-lactamases, which are bacterial enzymes that can hydrolyze β-lactam antibiotics, leading to bacterial resistance against these antibiotics. The inhibition mechanism is based upon the non-covalent competitive binding of BLIP to β-lactamases. The concave-shaped BLIP embraces β-lactamase by inserting its β hairpin loops into the active site of β-lactamase, completely masking β-lactamase’s active site from binding and hydrolyzing the β-lactam substrates (Strynadka et al. 1996). BLIP shows differential binding affinity to and inhibitory effect on different β-lactamases (Strynadka et al. 1994). It generally demonstrates specificity toward class A β-lactamases, inactivating them with inhibition constant (Ki) ranged from picomolar to micromolar (Doran et al. 1990; Rudgers et al. 2001; Zhang and Palzkill 2003; Yuan et al. 2011). In particular, it shows a potent inhibition against the clinically important TEM-1 β-lactamase with a Ki of 0.1–0.6 nM (Strynadka et al. 1994; Petrosino et al. 1999; Rudgers et al. 2001). On the contrary, BLIP does not inhibit class B, C and D β-lactamases (Strynadka et al. 1994).

Considering the tight interaction between BLIP and various class A β-lactamases, BLIP is an intriguing protein not only having its importance as a study model for protein–protein interaction but also showing its potential applications in biopharmaceutical industry and biotechnology. To fulfill the needs for sufficient supply of BLIP for various purposes, it is necessary to develop highly productive system to obtain BLIP.




Leave a Reply

Your email address will not be published.