Soluble expression of active recombinant human tissue plasminogen activator derivative (K2S) in Escherichia coli.

CONTEXT: The kringle 2 plus serine protease domains (K2S) of human tissue plasminogen activator (tPA) is an efficacious thrombolytic drug, which has been used to treat heart attacks and strokes by breaking up the clots that cause them. It has nine disulfide bridges, which are needed for proper folding and be the bottleneck in improving the production in the Escherichia coli system. So far, few reports have described the production of soluble active K2S from E. coli. OBJECTIVE: To achieve high-level expression of active K2S in the E. coli system. MATERIALS AND METHODS: The DNA fragment coding for K2S was fused with the E. coli disulfide isomerase DsbC. The constructed fusion protein was expressed in E. coli, and then purified with the Ni(2+)-chelating affinity chromatography. K2S was released by cleavage with Factor Xa protease, and the thrombolytic activity was determined using the fibrin plate assay. RESULTS: The fusion protein DsbC-K2S was found in the culture supernatant of recombinant E. coli as a soluble form of ~40%. The result of fibrinolysis fibrin plate assay showed that the purified recombinant K2S exhibited significant fibrinolysis activity in vitro. DISCUSSION AND CONCLUSION: These works provided a novel approach for the production of active K2S in E. coli without the requirements of in vitro refolding process, and might establish a significant foundation for the following production of K2S.

High-level expression of the antimicrobial peptide plectasin in Escherichia coli.

Plectasin is a defensin-like antimicrobial peptide isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. Plectasin showed marked antibacterial activity in vitro against Gram-positive bacteria, especially Streptococcus pneumoniae, including strains resistant to conventional antibiotics. Plectasin could kill the sensitive strain as efficaciously as vancomycin and penicillin and without cytotoxic effects on mammalian cell viability. In order to establish a bacterium-based plectasin production system, in the present study, the coding sequence of plectasin was optimized, and then cloned into pET32a (+) vector and expressed as a thioredoxin (Trx) fusion protein in Escherichia coli. The soluble fusion protein collected from the supernatant of the cell lysate was separated by Ni(2+)-chelating affinity chromatography. The purified protein was then cleaved by Factor Xa protease to release mature plectasin. Final purification was achieved by Ni(2+)-chelating chromatography again. The recombinant plectasin exhibited the same antimicrobial activity as reported previously. This is the first study to describe the expression of plectasin in E. coli expression system, and these works might provide a significant foundation for the following production or study of plectasin, and contribute to the development and evolution of novel antimicrobial drugs in clinical applications.

Intrabullous Adhesion Pexia (IBAP) by Percutaneous Pulmonary Bulla Centesis: An Alternative for the Surgical Treatment of Giant Pulmonary Bulla (GPB).

Background and Objective: Most patients with giant pulmonary bulla (GPB) are treated by surgery; however, there is a subset for whom surgery is not a viable option, such as those with contraindications, or those unwilling to undergo operation. Therefore, an alternative minimally invasive method is desired for this subpopulation. The aim of this study was to explore an alternative procedure for treating GPB. Methods: This was a prospective, nonrandomized, single-arm, unblinded study evaluating the efficacy and safety of intrabulla adhesion pexia (IBAP) procedure in GPB patients. The study was conducted between December 2004 and April 2017. Results: There were 38 cases in 36 patients (33 males and 3 females) with the target GPB cavities varying in size (range, 10 cm × 7 cm × 5 cm to 15 cm × 8 cm × 30 cm (anteroposterior diameter × medial-lateral diameter × superoinferior diameter)). After IBAP treatment, the closure ratio of GPB in one month was 86.84% (33/38), while the dyspnea index significantly decreased from 4.11 ± 1.11 to 2.24 ± 1.15 (P < 0.01). In addition, the mean FEV1 (L) increased from 1.06 ± 0.73 to 1.57 ± 1.13 (P < 0.01), while RV (L) decreased from 2.77 ± 0.54 to 2.36 ± 0.38 (P < 0.01) and TLC (L) decreased from 6.46 ± 1.21 to 5.86 ± 1.08 (P < 0.01). Moreover, PaO2 (mmHg) increased from 52.18 ± 8.31 to 68.29 ± 12.34, while the 6 MWD increased by 129.36% from 131.58 ± 105.24 to 301.79 ± 197.90 (P < 0.01). Collectively, these data indicated significant improvement in pulmonary function and exercise tolerance after IBAP treatment. Furthermore, no deaths occurred during IBAP treatment, and no cases of aggravated GPB relapse were reported during the 12-month follow-up period. Conclusions: IBAP is a promising strategy for the treatment of GPB. Our findings demonstrated that IBAP had a noteworthy therapeutic effect, desirable safety, and ideal long-term efficacy for GPB.