Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal XhoI site found at 124
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 433
Illegal AgeI site found at 1652
- 1000Illegal BsaI site found at 1655
This is a composite part for intracellular expression of FMO. Panb1 is a constitutive promoter in yeast, which is expressed under anaerobic conditions, while under aerobic conditions, Panb1, as a repression target of ROX1, is inhibited. When Panb1 initiates the expression, FMO is expressed and participates in the production from indole to indigo.
Usage and Biology
Flavin-containing monooxygenase, FMO is one kind of microsomal enzyme widely found in ER of most tissue, whose activity relies on flavin adenine dinucleotide (FAD), reduced nicotinamide adenine dinucleotide phosphate (NADPH) and oxygen. FMO could catalyze the oxidation of most exogenous compounds containing Nitrogen, Sulphur, Phosphorus, Selenium and other nucleophilic elements, which is a vital detoxifying process of carcinogen and many other hazardous substances. FMO is a kind of monooxygenase, which could produce C4a peroxyflavin intermediate, which is highly stable due to spectrum observation and could remain unchanged under 4℃ for minutes, even hours. In the first cycle, NADPH reduces FAD into FADH2. This reduced flavin could react with oxygen rapidly and become peroxyflavin, which is the main form of FMO and further react wih an appropriate nucleophilic substrate. This reaction will form one molecular of water and transfer one oxygen atom to the substrate. The release of NADP+ may be the rate-limiting step of catalytic cycle while the Vmax of substrate binding has little impact on both reactions.
Not quite to what we expect, after repeated transfection to the yeast, only a few products are expressed inside of eukaryotic system. Because of the large molecular weight and various types of some of our protein, we suspect that the common signal peptide we use, α-factor, is not enough to bring our protein out of the cell. While there is some of the genes without detectable products and we are hoping to get higher expression level, new primers for PCR are designed to ignore α-factor from our target gene in PCR. Then, likewise, we reconstruct this series of plasmid without α-factor through similar double-enzyme digestion and reconnection which insert our target genes right behind Panb1 promoter.
The bands of Panb1-crtB-AOX1 Terminator (less than 2000bp), Panb1-crtE-AOX1 Terminator (less than 2000bp) and Panb1-FMO-AOX1 Terminator (2500bp) from colony PCR are identical to the theoretical lengths of 1859bp, 1838bp and 2437bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid are successfully constructed.
To solve this, we reconstruct plasmids without the signal peptide and try to do intracellular expression. This is aim at all the undetectable or low-expressed genes.
The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast. Target genes are confirmed exist in the yeast of multiple bands, which could be the result of polluted electroporation cup.
After verification of successful transfection, we can’t test the protein directly due to intracellular expression. So, we extract the total protein in yeast and go for a purification through Nickel-affinity chromatography column, then apply SDS-PAGE to separate target protein from the large amount and various type of total protein to confirm whether our target protein could be expressed and value its expression level quantitatively.
Different from impure or permeate bands, the target protein located around 60kDa, bigger than the theoretical 53.96kDa but still within explainable and acceptable range of glycosylation modification. FMO could be confirmed as successfully expressed. The concentration of yeast total protein is so high that huge amount of impure protein is included during elution. But due to difference from impure or permeate bands, its dark color and consistency among several times of elution, this band could be verified as our target FMO,