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.

Prophenoloxidases 1 and 2 from the spruce budworm, Choristoneura fumiferana: molecular cloning and assessment of transcriptional regulation by a polydnavirus.

Immune challenge in arthropods is frequently accompanied by melanization of the hemolymph, a reaction triggered by the activation of prophenoloxidase (PPO). Because their immature stages are spent inside the hemocoel of insect larvae, endoparasitoids have evolved strategies to escape or counter melanin formation. Very little molecular information is available on these endoparasitoid counterstrategies. We have sought to shed light on the inhibition of melanization in the spruce budworm, Choristoneura fumiferana, by the parasitic wasp Tranosema rostrale, by cloning two host PPO homologs and studying their transcriptional regulation after parasitization. The two polypeptides are encoded by transcripts of approximately 3.3 kb (for CfPPO1) and 3.0 kb (for CfPPO2) and possess structural features typical of other insect PPOs. While there appears to be a single CfPPO2 gene in the C. fumiferana genome, we detected three CfPPO1 mRNA variants displaying insertions/deletions in the 3' untranslated region, suggesting that there may be more than one CfPPO1 gene copy. Both CfPPO1 and CfPPO2 were expressed at high levels in C. fumiferana 6th instars, and parasitization by T. rostrale had no apparent impact on the level of their transcripts. Injection of a large dose (0.5 female-equivalent) of polydnavirus-laden calyx fluid extracted from T. rostrale, which is known to inhibit melanization in C. fumiferana, only caused a transient decrease in CfPPO1 and CfPPO2 transcript accumulation at 2-3 d post injection. It thus appears that transcriptional downregulation of C. fumiferana PPO by T. rostrale plays a minor role in the inhibition of hemolymph melanization in this host-parasitoid system.

Assessment of promoter elements of the germ cell-specific proacrosin gene.

The testis-specific proacrosin gene encodes for a fertilization-promoting protein. In mouse and rat it is first transcribed in late pachytene spermatocytes and revealed to be translationally regulated. Former proacrosin promoter studies demonstrated that elements necessary for conducting a stage and temporal-specific expression of the gene are located within 0.9 kb upstream of the translational start codon. In the present study we analyzed putative cis-acting elements located in this promoter region for their specific binding properties to nuclear factors assumed to be involved in proacrosin gene regulation. Supplement of specific antibodies in electrophoretic mobility shift assays (EMSA) revealed that two Y-box proteins and the transcription factors CREM and YY1 interact with proacrosin promoter elements. The Y-box proteins, antigenically related to the frog Y-box proteins FRGY1 and FRGY2, bound to the Y-box (55-66 bp upstream of the ATG initiation codon) in brain and testis nuclear extracts, respectively. CREM bound to three elements (30-37, 252-259, and 717-724 bp upstream of ATG). The ubiquitous transcription factor YY1 bound to a conserved element in the central proacrosin promoter (457-473 bp upstream of ATG) and showed almost germ cell-specific truncates in EMSA. These results suggest that the identified factors are involved in proacrosin gene regulation.