Part:BBa_K4909001

elovl5

New Best Basic Part: BBa_K4909001 - elovl5

Profile

• Name: elovl5
• Base Pairs: 981bp
• Origin: Synthetic
• Properties: This gene belongs to the ELO family. It is highly expressed in the adrenal gland and testis, and encodes a multi-pass membrane protein that is localized in the endoplasmic reticulum. This protein is involved in the elongation of long-chain polyunsaturated fatty acids.

Usage and Biology

This gene belongs to the ELO family. It is highly expressed in the adrenal gland and testis, and encodes a multi-pass membrane protein that is localized in the endoplasmic reticulum. This protein is involved in the elongation of long-chain polyunsaturated fatty acids. Mutations in this gene have been associated with spinocerebellar ataxia-38 (SCA38). Alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Sep 2014].

A class of fatty acid carbon chain elongation enzymes, key enzymes involved in the synthesis of C18-C22 PUFAs, catalyse the introduction of two carbon atoms from the donor into the carbon chain of PUFAs to increase their carbon chain length. Biological membrane lipids and storage lipids both contain fatty acids of specific composition, and the differences in their chain lengths are controlled by fatty acid elongases that catalyse different chain length extension reactions. The mammalian ELOVL gene family is highly conserved and the expression product is a microsomal enzyme [1]. In this gene family, Elovl5 is involved in extending the carbon chains of C16 and C18 monounsaturated fatty acids (MUFAs) and C18, C20, and C22 PUFAs, and is a Highly unsaturated fatty acids (HUFAs) HUFAs), a key enzyme in the biosynthetic pathway [2-3].

Cultivation, Purification, and Identification

Construction of the pET-28a-elovl5 plasmid: The company synthesized the elovl5 gene's CDS sequence. Primer-assisted codon-optimized elovl5 CDS amplified by PCR. The target gene and pET-28a were then digested with HindIII/NotI, recovered, and purified. The target gene and vector were then ligated and transformed with T4 DNA ligase. The following day, recombinant plasmids were extracted, enzyme-digested to identify them, and positive clones were selected, amplified, and removed. The sequencing business received the positive recombinant plasmids for additional sequencing identification.

Figure 1: The structure of designed plasmids: pET-28a-ELOVL5

Figure 2: TAE agarose gel electrophoresis to verify the PCR outcomes

Figure 3: pET28-evovl5 sequencing results show it is successful.

Plasmid pET-28a-ELOVL5 was transfected into E. coli BL21 strain which is commonly used in plasmids transformation, and then to verify the protein expression in it. We induced the expression of proteins with IPTG when the OD600 was around 0.6-1.0, and cultured at 16°C for 12h. Subsequently, we used nickel affinity purification to purify the acquired proteins from other proteins in E. coli.

The protein yield calculated from nanodrop. ELOVL5 protein expression level is 3 mg/L. To test ELOVL5 proteins, we ran SDS-PAGE electrophoresis gel using FT, wash, and elution. The result showed that ELOV5 protein is found on elution1-3, marker 55KDa, indicating pET-28a-ELOVL5 is purified from E. coli BL21.

Figure 4: Expression and purification of protein ELOVL5.

Characterization/Measurement

We conducted an enzyme activity assay on the purified Elovl5 to validate its ability to elongate EPA into DPA. The experimental design was as follows: we established experimental and control groups, with the experimental group containing the strain Pet28a-Elovl5-BL21 for Elovl5 expression and purification, while the control group did not contain Elovl5. Samples were taken at various time points, including 0h, 8h, 24h, 48h, and 72h. According to the national standard method (GB5009.168-2016), the docosapentaenoic acid (DPA) in the fermentation broth was extracted and determined. The standard sample was dissolved in n-hexane (Sigma) and then directly loaded.

Determination of standard substance

According to Figure 5, the concentration of the standard is 0.5 mg / mL, and the area of the standard map is 633614752.

Figure 5: Determination of standard substance

Sample data

The docosapentaenoic acid (DPA) content was detected by adding pET28a-elovl5 (BL21) bacteria at 37 °C to detect the activity of elovl5. The pET28a (BL21) was used as a blank control. Table 1 shows the peak area and concentration of samples and controls.

It can be seen that Figure 6A does not have the peak area of the docosapentaenoic acid (DPA), Figure 6B does not have the peak area of the docosapentaenoic acid (DPA), Figure 6C has a clear peak of the docosapentaenoic acid (DPA), with an area of 1425052, and Figure 6D has a clear peak of the docosapentaenoic acid (DPA), with a peak area of 2264369. Therefore, it can be inferred that elovl5 catalyzes the formation of docosapentaenoic acid (DPA). The enzyme elovl5 has activity.

Figure 6: The peak area of the docosapentaenoic acid (DPA)
Note: A: the docosapentaenoic acid (DPA) of pET28a-elovl5(BL21)-0h. B: the docosapentaenoic acid (DPA) of pET28a(BL21). C: the docosapentaenoic acid (DPA) of pET28a-elovl5(BL21)-12h D: the docosapentaenoic acid (DPA) of pET28a-elovl5(BL21)-24h

References

1. Zank TK, Zähringer U, Beckmann C, et al. Cloning and functional characterisation of an enzyme involved in the elongation of Δ6-polyunsaturated fatty acids from the moss Physcomitrella patens. Plant J, 2002, 31(3): 255-268.
2. Vasconcelles MJ, Jiang YD, McDaid K, et al. Identification and characterization of a low oxygen response element involved in the hypoxic induction of a family of Saccharomyces cerevisiae genes: implications for the conservation of oxygen sensing in eukaryotes. J Biol Chem, 2001, 276(17): 14374-14384.
3. Sprecher H. Metabolism of highly unsaturated n-3 and n-6 fatty acids. Biochim Biophys Acta Mol Cell Biol Lipids, 2000, 1486(2/3): 219-231.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 924
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]