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.

Efficient expression and secretion of recombinant human growth hormone in the methylotrophic yeast Pichia pastoris: potential applications for other proteins.

A simple high yielding process for the production of rhGH (recombinant human growth hormone) in the Pichia pastoris system is described. The approach adopted the addition of surfactants during fermentation along with methanol induction. A Pichia integrant harbouring multiple-copy, non-codon-optimized hGH showed poor expression in complex and defined media. Inclusion of the surfactants Tween 20 or Tween 80 during induction enhanced the expression levels significantly in shake flask studies. The combination of 2 litres of basal salt medium and Tween 20 in a bioreactor culminated in 3 x 10(4)-fold elevated expression of the protein (approximately 500 mg/l) as estimated by ELISA. SDS/PAGE and Western-blot analyses revealed that the Pichia-derived rhGH migrated as a single band with a molecular mass of approximately 22 kDa and had the same immunoreactivity as native commercial rhGH. Analysis of Pichia-derived purified rhGH and commercial rhGH on an Agilent 2100 Bioanalyzer revealed overlapping peaks displaying authentic N-terminal processing of Pichia-derived rhGH, which was further confirmed by N-terminal sequencing. In addition, matrix-assisted laser-desorption ionization-time of flight analysis of the protein confirmed its authenticity. These results indicate that the P. pastoris expression system can be effective in the production of rhGH at commercially relevant levels.

Yeast extract mediated autoinduction of lacUV5 promoter: an insight.

We report a simple and cost-effective autoinducible media component responsible for the autoinduction of proteins in Escherichia coli under lacUV5 promoter system. Yeast extract (YE) at high concentration was found to stimulate the expression of T7 RNA polymerase in BL21(DE3) cells while such an effect was not seen in BL21A1 cells. A systematic study on the effect of varying concentrations of YE indicated several folds higher expression of genes viz., human granulocyte colony stimulating factor (rhGCSF), human interferon alpha 2b (rhIFN-alpha2b) and Staphylokinase (rSAK) in BL21(DE3) cells in the absence of any specific inducer like IPTG or additional lactose. Additional investigations on the inducible component of the YE revealed the presence of significant amount of endogenous lactose as the contributory factor for the observed autoinduction phenomenon. This paper highlights the easy scalability of the use of the present media component for large-scale production in biotechnology industry.