Screening Of β-lactamase Inhibitors By Yeast Two-hybrid System

β-lactam antibiotics (penicillin, cephalosporin, and their semi-synthetic derivatives) are used to treat various bacterial infections for clinical purpose. They have made great contribution to humans' health. But as following widespread use of antibiotics and abuse to some extends, drug resistance bacteria were emerged gradually. Many bacteria are resistant to β-lactam antibiotics, thereby those drugs could not cure diseases which were brought by some drug resistance bacteia. Clavulanic acid, a β-lactamase inhibitor obtained from microbe, and compounds of β-lactam sulfone, such as sulbactam and tazobactam, were found and demonstrated to inhibit β-lactamase in 1970s and 1980s. Each was coupled with a β-lactam antibiotic to improve the potency and longevity of the partner drug. Unfortunately, those inhibitors could not inhibit TEM-1 β-lactamase mediated by chromosome to great extend. Recently, BLIP (β-lactamase inhibitory protein) was isolated from Streptomyces clavuligerus. Advanced researches indicated that the residues of 46 to51 of BLIP made critical interactions with the active site of TEM-1 β-lactamase. The new type proteinaceous β-lactamase inhibitor brought many prospect to scientists. Studies of BLIP were payed a lot of attention. In this research, we expected that small peptides which were able to inhibit β-lactamase can be screened from this random nucleotide library by yeast two hybrid system. A random oligonucleotide fragment was designed and some important information about BLIP interacting β-lactamase was designed into this random fragment. One hand it could extend spectrum of β-lactamase inhibitory peptide, the other hand it could enhance probability of screening. Researches as following:Firstly, higher homogeneous β-lactamase was needed to test peptide inhibitory activity gained by yeast two hybrid screening in this research. So β-lactamase gene was subcloned into vector pLY-5 and plasmid pYG201 was constructed. In plasmid pYG201, β-lactamase was expressed under the regulation of P_l promoter from λ phage, β-lactamase was expressed high efficiently by 42 ℃ induction which could relieve repression β-lactamase was expressed in the form of inclusion body, then the processes for wash, denaturation, refolding and purification of inclusion body were studied. Finally β-lactamase was purified up to 90% homogeneity.Random single chain oligo nucleotides were amplified to doublechain nucleotides by PCR method, then double-chain nucleotides were fused to active domain of pGAD424 by a series of molecular biology manipulation and random nucleotide library (prey plasmid library) was constructed. The optimum conditions for yeast transformation were determined as PEG4000 was used as accelerant and 30min for heat shock time. Transformation efficiencywas 1.4 X 105cfu/ugDNA under this conditions. So the following yeast two hybrid screening was based on those studies.Plasmid pYGlll and pYG202 (constructed random nucleotide library) were transformed into Saccharomyces cerevisiae Y153. three positive transformants were gained by screening and re-screening, plasmids were isolated from those positive transformants and named pYG202-pl, pYG202-p2, and pYG202-p3, respectively. Quantitative assay of P-galactosidase activity indicated that the interaction between P3 and P-lactamase in vivo was the stronges that P3 has the strongest interaction with p-lactamase in vivo, its Miller units was 10.2, whereas PI and P2 Miller units were 4.3 and 2.1, respectively. Sequence analysis suggested that there was larger difference among these random sequence of in these three positive transformants. But three peptides encoded by their gene sequence could all interact with P-lactamase in vivo. The results suggest core amino acid sequence (AAGDYY) may play a critical role provided critical function.In this work, DNA fragments coding for PI and P3 which can bind P-lactamase were obtained from a random DNA fragment bank by yeast two-hybrid system screening, and then subcloned into pGEX-4T-l, and therefore, recombinant plasmids p...