Expression and purification of soluble recombinant ß-lactamases using Escherichia coli as expression host and pET-28a as cloning vector.

BACKGROUND: Due to its high expression capability, recombination of Escherichia coli and pET vector has become the bioengineering preferred expression system. Because ß-lactamases mediate bacterial antimicrobial resistance, these enzymes have a substantial clinical impact. Using the E. coli expression system, several kinds of ß-lactamases have been produced. However, previous studies have been focused on characterizing target ß-lactamases, and the effects of cultivation and induction conditions on the expression efficiency of target enzymes were not addressed. RESULTS: Using pET-28a as the cloning vector and E. coli BL21(DE3) as the expression host, this study originally elucidated the effects of IPTG concentration, culture temperature, induction time, and restriction sites on recombinant ß-lactamase expression. Moreover, the effects of the target protein length and the 6 × His-tag fusion position on enzyme purification were also explored, and consequently, this study yielded several important findings. (i) Only the signal peptide-detached recombinant ß-lactamase could exist in a soluble form. (ii) Low-temperature induction was beneficial for soluble ß-lactamase expression. (iii) The closer to the rbs the selected restriction site was, the more difficult it was to express soluble ß-lactamase. (iv) The short-chain recombinant protein and the protein with His-tag fused at its C-terminus showed high affinity to the Ni2+ column. CONCLUSIONS: Based on our findings, researchers can easily design an effective program for the high production of soluble recombinant ß-lactamases to facilitate other related studies.

[Kneading and dispersing manipulation in treatment of early-stage acute mastitis: a randomized controlled trial].

BACKGROUND: Acute mastitis is an acute infectious disease of breast. Antibiotic treatment is often unable to obtain a good effect, and we should actively look for a safe and effective non-drug therapy. OBJECTIVE: To validate the clinical efficacy of kneading and dispersing manipulation in treatment of early-stage acute mastitis. DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: According to the multicenter randomized controlled trial design, 198 cases of acute mastitis from Yueyang Hospital of Integrated Traditional Chinese Medicine, Longhua Hospital, and Shanghai Yangpu Maternity and Child Health Hospital were randomly divided into treatment group and control group. There were 99 cases in each group. Patients in the treatment group were only treated with manipulation, and cefradine was orally administered to patients in the control group. MAIN OUTCOME MEASURES: The local breast lump size, clinical symptoms and the adverse reactions in the two groups were observed before and after the treatment. RESULTS: The total response rates in the treatment and control group were 95.92% (94/98) and 80% (76/95) respectively. There was a significant difference in the total response rate between the two groups (P<0.05). There were significant differences in the score of breast lump size, and the score of signs and symptoms between the two groups (P<0.05). CONCLUSION: Kneading and dispersing manipulation has certain effects on early-stage acute mastitis, and the therapy is safe and repeatable.