Enhanced fluorescent properties of an OmpT site deleted mutant of green fluorescent protein.

BACKGROUND: The green fluorescent protein has revolutionized many areas of cell biology and biotechnology since it is widely used in determining gene expression and for localization of protein expression. Expression of recombinant GFP in E. coli K12 host from pBAD24M-GFP construct upon arabinose induction was significantly lower than that seen in E. coli B cells with higher expression at 30 degrees C as compared to 37 degrees C in E. coli K12 hosts. Since OmpT levels are higher at 37 degrees C than at 30 degrees C, it prompted us to modify the OmpT proteolytic sites of GFP and examine such an effect on GFP expression and fluorescence. Upon modification of one of the two putative OmpT cleavage sites of GFP, we observed several folds enhanced fluorescence of GFP as compared to unmodified GFPuv (Wild Type-WT). The western blot studies of the WT and the SDM II GFP mutant using anti-GFP antibody showed prominent degradation of GFP with negligible degradation in case of SDM II GFP mutant while no such degradation of GFP was seen for both the clones when expressed in BL21 cells. The SDM II GFP mutant also showed enhanced GFP fluorescence in other E. coli K12 OmpT hosts like E. coli JM109 and LE 392 in comparison to WT GFPuv. Inclusion of an OmpT inhibitor, like zinc with WT GFP lysate expressed from an E. coli K12 host was found to reduce degradation of GFP fluorescence by two fold. RESULTS: We describe the construction of two GFP variants with modified putative OmpT proteolytic sites by site directed mutagenesis (SDM). Such modified genes upon arabinose induction exhibited varied degrees of GFP fluorescence. While the mutation of K79G/R80A (SDM I) resulted in dramatic loss of fluorescence activity, the modification of K214A/R215A (SDM II) resulted in four fold enhanced fluorescence of GFP. CONCLUSIONS: This is the first report on effect of OmpT protease site modification on GFP fluorescence. The wild type and the GFP variants showed similar growth profile in bioreactor studies with similar amounts of recombinant GFP expressed in the soluble fraction of the cell. Our observations on higher levels of fluorescence of SDM II GFP mutant over native GFPuv in an OmpT+ host like DH5alpha, JM109 and LE392 at 37 degrees C reiterates the role played by host OmpT in determining differences in fluorescent property of the expressed GFP. Both the WT GFP and the SDM II GFP plasmids in E. coli BL21 cells showed similar expression levels and similar GFP fluorescent activity at 37 degrees C. This result substantiates our hypothesis that OmpT protease could be a possible factor responsible for reducing the expression of GFP at 37 degrees C for WT GFP clone in K12 hosts like DH5alpha, JM109, LE 392 since the levels of GFP expression of SDM II clone in such cells at 37 degrees C is higher than that seen with WT GFP clone at the same temperature.

Strategies to maximize expression of rightly processed human interferon alpha2b in Pichia pastoris.

The human interferon alpha 2b (IFN alpha2b) belongs to the interferon family of cytokines that exerts many biological functions like inhibition of virus multiplication, repression of tumour growth and other immunological functions. Herein, a synthetic gene coding for human IFN alpha2b was cloned and integrated into a methylotropic yeast-Pichiapastoris. The recombinant human IFN alpha2b protein (approximately 19kDa) could be successfully expressed in Pichiapastoris to a level of nearly 300mg/L with nearly 93% recovery on purification using a single anion exchange chromatography step. A novel media component dimethyl sulphoxide (DMSO) was found to aid in expression of rightly processed IFN alpha2b form with dramatic reduction in the expression of a 20kDa IFN isoform contaminant frequently observed by other workers. The identity of the 20kDa isoform was confirmed by N terminal sequencing which showed extra eleven amino acids at the N terminal portion of the IFN molecule obtained due to incorrect processing by the host KEX2 protease. The purified IFN alpha2b (19kDa) preparation was confirmed by N terminal sequencing, and characterized by MALDI-TOF and Agilent 2100 Bioanalyzer. The bioassay of the recombinant protein gave a specific activity of >2x10(8)IU/mg.

Novel self-cleavage activity of Staphylokinase fusion proteins: An interesting finding and its possible applications.

Staphylokinase (SAK) is reported to have a serine protease domain with no proteolytic activity unlike other plasminogen activators like tissue plasminogen activator (t-PA) and urokinase. A unique protease property of Staphylokinase was observed when SAK was expressed as a fusion protein in inducible Escherichia coli expression vectors. This finding was further investigated by cloning and expressing different SAK fusions, both native and N-terminal deletions, with fusion tags like glutathione S-transferase (GST) and signal sequence of SAK in bacterial system. While all the N-terminal SAK fusions were found to self-cleave in crude and purified preparations, the C-terminal SAK fusion was stable. The cleavage property of Staphylokinase fusion proteins, inhibited by reduced glutathione and PMSF, was independent of its thrombolytic activity and also independent on the type of host employed for its expression. The serine protease domain of the SAK gene possibly lies between 20th to 77th amino acid and serine 41 of this region appears critical for such a cleavage property.

Over-expression of proteins using a modified pBAD24 vector in E. coli expression system.

A modified pBAD24 vector (pBAD24M) was constructed with the araBAD promoter of the arabinose operon along with T7g10 sequence elements and a modified Shine-Dalgarno sequence. While both green fluorescent protein and granulocyte colony stimulating factor showed negligible expression under the original pBAD24 vector, they were expressed at >35% of total cellular protein with the modified vector. Similar results were obtained for staphylokinase wherein the pBAD24-SAK construct yielded 8 ng/10(6) c.f.u. of E. coli induced cells while the pBAD24M-SAK vector showed nearly 55 ng/10(6) c.f.u. induced bacterial cells as tested by ELISA. Interestingly, the expression levels using modified pBAD24 vector matched that achieved with T7 promoter based vector system. The modified pBAD24 vector therefore represents a simple and a useful prokaryotic expression system for efficient repression, modulation and elevated protein expression levels.

Effective protection by high efficiency bicistronic DNA vaccine against infectious bursal disease virus expressing VP2 protein and chicken IL-2.

Infectious bursal disease (IBD) is an acute and contagious viral infection of young chickens caused by IBD virus (IBDV). IBDV belongs to genus Avibirnavirus of family Birnaviridae. It is a non-enveloped virus with icosahedral symmetry that contains two segments of double-stranded RNA. The virus affects the lymphoid tissues of chickens, mainly the B cells of bursa of Fabricius, leading to severe and prolonged immunosuppression. VP2, a major structural protein of IBDV, contains antigenic epitopes responsible for induction of neutralizing/protective antibody. In the present study, VP2 gene of IBDV was cloned in a bicistronic vector along with chicken interleukin-2 (chiIL-2) as an adjuvant. An in vivo challenge study of bicistronic DNA vaccine expressing IBDV-VP2 and chicken IL-2 showed effective protection against a lethal IBD infection in chickens. In addition, mortality, gross picture of bursa and histopathological findings demonstrated the efficacy of the vaccine in reducing virulence of the disease.