Highly efficient recombinant production and purification of streptococcal cysteine protease streptopain with increased enzymatic activity.

Streptococcus pyogenes produces the cysteine protease streptopain (SpeB) as a critical virulence factor for pathogenesis. Despite having first been described seventy years ago, this protease still holds mysteries which are being investigated today. Streptopain can cleave a wide range of human proteins, including immunoglobulins, the complement activation system, chemokines, and structural proteins. Due to the broad activity of streptopain, it has been challenging to elucidate the functional results of its action and precise mechanisms for its contribution to S. pyogenes pathogenesis. To better study streptopain, several expression and purification schemes have been developed. These methods originally involved isolation from S. pyogenes culture but were more recently expanded to include recombinant Escherichia coli expression systems. While substantially easier to implement, the latter recombinant approach can prove challenging to reproduce, often resulting in mostly insoluble protein and poor purification yields. After extensive optimization of a wide range of expression and purification conditions, we applied the autoinduction method of protein expression and developed a two-step column purification scheme that reliably produces large amounts of purified soluble and highly active streptopain. This method reproducibly yielded 3 mg of streptopain from 50 mL of expression culture at >95% purity, with an activity of 5306 ± 315 U/mg, and no remaining affinity tags or artifacts from recombinant expression. This improved method therefore enables the facile production of the important virulence factor streptopain at higher yields, with no purification scars that might bias functional studies, and with an 8.1-fold increased enzymatic activity compared to previously described procedures.

Treatment of severe vertebral body compression fractures with percutaneous vertebroplasty.

OBJECTIVE: Evaluate the efficacy of percutaneous vertebroplasty for severe vertebral body compression fractures. METHODS: Over a period of 6 years and 8 months, 661 vertebroplasties were performed in 292 patients at our institution. Of these, 69 patients met our criteria for a severe vertebral body compression fracture defined as vertebral body collapse to less than one-third of the original height. Of the 69, 25 underwent single level vertebroplasty. Imaging features were then analyzed including location, extent of collapse, pattern of compression, pre- and post-kyphotic angle and adjacent disc height. Complications and clinical outcomes were then evaluated. RESULTS: Involved vertebra ranged from T6 to L5 with 60% at the thoracolumbar junction. Vertebral body collapse ranged from 30 to 14% (mean 22%) of original height. Pattern of collapse included 11/ 25 (44%) plana, 8/25 (32%) gibbus, and 6/25 (24%) H-shaped. Kyphotic angle before vertebroplasty ranged from 33-0° (mean 16°) with an average correction of 1.2° after vertebroplasty. Mean disc height before vertebroplasty was 7.3 mm above and 7.7 mm below. Complications included cement leak to the adjacent disc in 16 (64%) and the paravertebral soft tissues in 3 (12%). Cement leak into the proximal azygous vein was documented in one case. International Quality of Life Questionnaire VAS was completed before and after (6 weeks) the procedure by all but six patients. Mean pre-intervention VAS was reported as 7.00 (range 5-10, SD 1.73) and mean post-intervention VAS was reported as 5.11 (range 0-9, SD 2.56), demonstrating a statistically significant improvement in pain (P < 0.015, 95% CI = 0.83-2.96) with 84% or 16/19 patients reporting some degree of improvement. CONCLUSION: Percutaneous vertebroplasty is safe and effective in the treatment of single level severe vertebral body compression fractures.

Magnetic resonance imaging appearance of partial latissimus dorsi muscle tendon tear.

There is still a paucity of information about the clinical presentation, treatment and imaging findings of latissimus muscle tears. Only one study has specifically described the magnetic resonance imaging (MRI) features of latissimus tendon tears. We describe a case of a high-grade tear in the latissimus muscle tendon in an active water skier with no significant prior medical history. MRI demonstrated at least a 50% tear of the latissimus tendon, manifesting as increased signal intensity on T2-weighted sequences and surrounding edema, as well as a diminutive tendon at the humeral insertion.

Advances in the directed evolution of proteins.

Natural evolution has produced a great diversity of proteins that can be harnessed for numerous applications in biotechnology and pharmaceutical science. Commonly, specific applications require proteins to be tailored by protein engineering. Directed evolution is a type of protein engineering that yields proteins with the desired properties under well-defined conditions and in a practical time frame. While directed evolution has been employed for decades, recent creative developments enable the generation of proteins with previously inaccessible properties. Novel selection strategies, faster techniques, the inclusion of unnatural amino acids or modifications, and the symbiosis of rational design approaches and directed evolution continue to advance protein engineering.