Expression and Purification of Matrix Metalloproteinases in Escherichia coli.

The MMP (matrix metalloproteinases) family of endopeptidases are involved in cleavage induced remodelling of the extracellular matrix including collagen, fibrinogen, elastin, and gelatin. Owing to their proteolytic activity which can cleave and degrade multiple intracellular substrates, the overexpression and purification of these proteins tends to be toxic. Here we describe a novel "matrix assisted refolding" protocol to overcome the technical challenges associated with overexpression and purification of full-length MMPs. The toxicity issue associated with MMP expression, is circumvented by expressing the recombinant protein in Escherichia coli in an inactive insoluble form. The methodology used for obtaining full-length MMP2 protein from these inclusion bodies, by its subsequent purification and refolding using affinity chromatography, through a single-step matrix based refolding protocol is presented here. The protocol described yields high concentrations of pure full-length and active MMP2 protein useful for downstream applications.

FRET reveals multiple interaction states between two component signalling system proteins of M. tuberculosis.

BACKGROUND: Two component signalling involves interaction between sensor kinase (SK) and response regulator (RR) proteins which depends on their phosphorylation status. METHODS: In this study we report the development of an in vitro FRET assay for studying interaction between fluorescently tagged SK and RR proteins. RESULTS: Using TCS proteins of Mycobacterium tuberculosis, we demonstrate that phosphorylation status of SK affects the SK-RR interaction, which varies from one TCS to another. The observation was strengthened by recordings from mutant SK and RR proteins. The assay retained the specificity/crosstalk potential of the participating proteins and reflected the inherent phosphotransfer potentials. CONCLUSIONS: SK and RR proteins interact with each other in unphosphorylated state and the phosphorylation affects the interaction between SK and RR, which was reflected as reduction in FRET ratio. GENERAL SIGNIFICANCE: A non-radioactive, in vitro FRET based assay is reported, which can be utilized for studying genome-wide partner screening, identifying crosstalk or specificity in TCSs.

Matrix-assisted refolding, purification and activity assessment using a 'form invariant' assay for matrix metalloproteinase 2 (MMP2).

Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed 'form invariant' assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol.

A case of flax seed induced rhabdomyolysis.

Rhabdomyolysis is a clinical and a biochemical syndrome that occurs due to a skeletal muscle injury. The main cause of rhabdomyolysis is a muscle crush injury, toxins, ischaemia and metabolic disorders. Rare cases of rhabdomyolysis have been reported which had been caused by drugs and after insect stings. The breakdown products of the damaged muscle cells are released into the bloodstream; some of these, such as the protein myoglobin, are harmful for the kidneys and they may lead to kidney failure. The symptoms of rhabdomyolysis depend on the severity of the condition. The milder forms of rhabdomyolysis may not cause any muscle symptoms, and the diagnosis is based on abnormal blood tests. The most reliable test in the diagnosis of rhabdomyolysis is the blood level of Creatine Kinase (CK) which is released by the damaged muscles. Here in, we report an unusual case of flax seed induced rhabdomyolysis to alert the medical community about this rare complication.

Evaluation of knowledge of and attitudes toward epilepsy among the health science students of Manipal University.

This study was carried out to assess the knowledge, attitudes, and practices with respect to epilepsy among health science students of Manipal University. The study was conducted among first-year health science students of Kasturba Medical College on the Manipal and Mangalore campuses of Manipal University. All students willing to participate (n=587) were asked to answer a standardized questionnaire on epilepsy. Our study group included educated urban people from various ethnic backgrounds of various countries who chose health science as a career, which makes the study different from others. Even though most of the students had heard about epilepsy, very few knew about the underlying cause. Most of the students were ignorant about the drugs and ways to deal with epileptic seizures. Questions related to community and religion were deliberately avoided as some subjects found such questions offensive, which may have resulted in a difference in response as compared with previously published studies. It was observed that a large number of students were not aware of the nuances of epilepsy. However, we expect the knowledge, attitudes and awareness to improve considerably with time and increased exposure. These findings support a need to promote epilepsy awareness programs as a means of increasing public knowledge of epilepsy.