The characterization, cloning and overexpression of BLIP gene in Pichia pastoris and Bacillus subtilis

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The characterization, cloning and overexpression of BLIP gene in Pichia pastoris and Bacillus subtilis

 

Author: Law, Kin-ho
Title: The characterization, cloning and overexpression of BLIP gene in Pichia pastoris and Bacillus subtilis
Degree: Ph.D.
Year: 2009
Subject: Hong Kong Polytechnic University -- Dissertations.
Pichia pastoris -- Genetics.
Bacillus subtilis -- Genetics.
Department: Dept. of Applied Biology and Chemical Technology
Pages: xxxii, 194 leaves : ill. ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2321040
URI: http://theses.lib.polyu.edu.hk/handle/200/5093
Abstract: The introduction of b-lactam antibiotics in clinical trials more than 60 years ago clearly represents one of the major breakthroughs in modern chemotherapy. Nowadays, b-lactams still account for approximately 60% of global antibiotic consumption. The production of one or more b-lactamases by pathogenic bacteria represents the most widespread resistance mechanism to antibiotics. The use of b-lactamase inhibitors has been shown to be more successful in recent years. These compounds structurally resemble penicillin, each demonstrates high affinity (nM -uM) for b-lactamases. b-Lactamase inhibitors work by acting as very slowly metabolized substrates, thereby occupying the active site long enough such that the antibiotics are not degraded before their antibacterial action has been completed. The b-lactamase inhibitory protein (BLIP) is a 165 amino acid protein produced by the Gram-positive bacterium Streptomyces clavuligerus. It has been shown to be a potent inhibitor of bacterial class A b-lactamases in many studies. In this study, heat-inducible and methanol inducible expression systems were developed for BLIP expression in Bacillus subtilis and Pichia pastoris respectively. Although BLIP has no antimicrobial activity on its own, this inhibitor is used in conjunction with various b-lactam antibiotics to bind b-lactamase and prevent the hydrolysis of the antibiotics. It can restore their therapeutic value and prolong the effectiveness of these antimicrobial agents. BLIP was not expressed in recombinant B. subtilis intracellularly or extracellularly in shake flasks. The BLIP gene cannot be functionally expressed in B. subtilis. However, the recombinant BLIP was successfully produced by Pichia pastoris X-33 and was secreted into the culture medium. The purity of this fully active BLIP was >80% even without any purification steps. The recombinant BLIP produced by Pichia pastoris KM71 was about half the amount of BLIP produced by Pichia pastoris X-33. We have developed an efficient production process in which concentrations of 2-3% of methanol and 30 oC during induction phase, produced the best results. A complex growth medium (BMGY) was chosen to give higher cell density and hence higher BLIP production. The total time of cultivation was similar to that of the native host S. clavuligerus, but an unprecedented high yield of recoverable protein in the culture supernatant (~300 mg of >80% pure BLIP/L culture) was achieved. The amount of recombinant BLIP produced by P. pastoris X-33 is ~20-fold higher than the ~15 mg/L produced by S. clavuligerus. Heterologous gene expression was lightly controlled and no production of BLIP was observed before the methanol-induction, suggesting that cell density can be further increased to improve protein yield. The amount of dissolved oxygen was found to be important for the recombinant BLIP expression in the Pichia expression system. Molecular weight of the P. pastoris-expressed BLIP measured by ESI-MS was 18212.72 +- 3.96, consistent with the calculated value. An inhibition constant (Ki values of 0.55 +- 0.07 nM (flask culture); 0.30 +- 0.06 nM (fed-batch fermenter culture) and 0.61 +- 0.12 nM (the continuous fermentation culture) were obtained for the interaction between BLIP and the E. coli TEM-1 b-lactamase, similar to the value (~0.6 nM) found for that of the native S. clavuligerus-produced BLIP. The total amount of BLIP produced was still low due to the limitations and lack of control in flask fermentations. Hence, the process was optimized in a fermenter. The growth of a recombinant Bacillus subtilis strain (with both PenP and PenPC beta-lactamase genes) was not inhibited by BLIP alone. However, in the presence of ampicillin, there was no observable growth of the Bacillus in all the cultures with 2.5 uM BLIP protein within 18 hours. This indicates that the BLIP in combinations with the b-lactam antibiotic -ampicillin was effective in killing the b-lactamase producing strain. The complex medium (BMGY) was used in fermenter experiments. Fed-batch culture strategy was used. Nitrogen nutrient such as tryptone was fed into the culture during induction and was found to be important for BLIP expression. P. pastoris X-33 was found to be superior for BLIP expression and more than 500 mg/L secreted proteins were found in the supernatant. However, the production of other cellular proteins of P. pastoris increased with the cell density and at the end of the fermentation constituted more than 50% of the total secreted proteins. DO-stat (feeding controlled by dissolved oxygen) and continuous feeding of the methanol during induction was compared, there were no significant difference in the amount of BLIP produced. Continuous culture strategy was studied. At dilution rate=0.015-0.02, ~100 mg/L of BLIP with ~80% purity was obtained in about 7 days after methanol induction. Although the fresh medium was continuously fed in and diluted the secreted BLIP in the medium, there was no significant decrease in the BLIP content from both the SDS-PAGE and the Bradford assay. The cells grew and secreted the BLIP protein continuously to compensate the loss from the culture removed from the fermenter during the continuous fermentation. Different protein chromatography methods were used in purifying the recombinant BLIP. These include hydrophobic interaction chromatography by phenyl HP column, ion-exchange chromatography by using Q-Sepharose column, DEAE-Sepharose column and SP column. However, results showed that purification of BLIP with these methods were not satisfactory. In another attempt to purify BLIP, recombinant histidine-tagged BLIP was sub-cloned and successfully expressed by P. pastoris. The BLIP contained histidine on the 3' end, and theoretically could be purified by nickel affinity chromatography. However, more than 50% of the BLIP did not bind to the affinity column. Purification protocol based on the tangential flow filtration (TFF) was finally developed and ~90% pure BLIP was obtained. To further test the ability of the P. pastoris in expressing different BLIP genes, mutant BLIP genes V3C and E73A/Y143A were successfully cloned and expressed in flask culture. Both mutated BLIP was functionally active when determined by spectrometric b-lactamase inhibition assay.

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