Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 1212
- 21Illegal BglII site found at 310
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 826
Illegal AgeI site found at 1503
- 1000Illegal BsaI.rc site found at 1292
Usage and Biology
Lipoxygenase (LOX) attaches oxygen to the acyl groups of polyunsaturated fatty acids or glycerides to form corresponding hydroperoxides. Lipoxygenase and peroxidase are involved in the degradation of lycopene in food, which requires the existence of oxygen and activating cofactors at the same time. Because lycopene is a compound of carotenoids, the biological function of β-carotene in carotenoids is attributed to its ability to scavenge free radicals and physical quenching of singlet oxygen. And produce vitamin A (retinol). Although lycopene can not be converted into vitamin A, it has a strong effect of scavenging singlet oxygen, scavenging free radicals and inhibiting lipid peroxidation. Lycopene has the strongest antioxidant effect among carotenoids, especially at twice the rate of β-carotene. In the process of lycopene degradation caused by lipoxygenase, lipoxygenase first catalyzes the oxidation of unsaturated or polyunsaturated fatty acids, and the resulting peroxide reacts with lycopene to promote the degradation of lycopene, in order to prevent the rancidity of oily food.
Plasmid with target gene is transformed into E.coli. From them, we acquire large amount of target gene using as raw material for further operation.
The bands of AOX1-α factor-FMO-AOX1 Terminator (3000+bp), AOX1-α factor-crtE-AOX1 Terminator (almost 3000bp), AOX1-α factor-crtB-AOX1 Terminator (less than 3000bp) and AOX1-α factor-crtI-AOX1 Terminator (3000+bp) from colony PCR are identical to the theoretical lengths of 3214bp, 2746bp, 2767bp and 3316 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid had successfully transformed into E.coli. Using E.coli for amplification, we extract and digest them with Bgl I or Sal I to get linear plasmid, which could be integrated into yeast genome to avoid getting lost while being frozen. Then, concentration of linear plasmid is also applied to achieve higher copy number and higher expression level. Several rounds of electroporation later, we successfully get all the plasmid with AOX1 as promoter into yeast.
After confirmation from colony PCR and sequencing, we using the successfully integrated yeast for expression. At first, we try to detect our target protein in the supernatant since there is signal peptide.
Due to glycosylation modification of yeast expression, the molecular weight exhibited on SDS-PAGE will be larger than theoretical. Primary detection shows that we have laccase, 4CL and ACC bands of about 75kDa, LOX2 band of 100+kDa and DsbC+pepACS of about 40kDa, all of which is a bit larger(Laccase：57.01 kDa; 4CL：61.88 kDa; ACC：63.40 kDa; LOX2：102.88 kDa; DsbC+pepACS：31.72 kDa) but still within explainable and acceptable range, which could be evidence of successful expression.
Enzyme activity determination
After the target strips appeared on SDS-PAGE, for the enzymes with standard enzyme activity assay such as Laccase and LOX2, we measured their enzyme activity according to the standard measurement methods to detect whether our enzymes are catalytically active. Laccase and LOX2 show high activity after adjusting the relevant conditions such as pH, temperature and ion concentration.
For LOX2: use 27ul linoleic acid, 25ul tween 20, 8ml ddH20, 3M/L NaOH 183ul, and dilute to 50ml as a solution; take 200ul substrate, 1ml supernatant, and 1ml pH6.0 buffer for measuring absorbance changes at 234nm. And every 0.001 increase in absorbance is regarded as an enzyme activity unit. Its activity was successfully detected.