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.
Salunkhe
Shardul S
SS
Lupin Limited, Biotechnology R & D, Mulshi Taluka, Pune, India.
Raiker
Veena A
VA
Rewanwar
Sachin
S
Kotwal
Prakash
P
Kumar
Avijeet
A
Padmanabhan
Sriram
S
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005453:Fluorescence; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C499588:ompT protein, E coli; D049452:Green Fluorescent Proteins; D010447:Peptide Hydrolases