Part:BBa_K3711015

AOX1

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

The Pichia Pastoris expressing system is one of the most popular hosts of external protein expression, and the most popular and effective promoter of it is alcohol oxidase1, AOX1 promoter, which is strongly induced by methanol while inhibited by carbon source like glucose, glycerol and ethanol. The regulation of AOX1 is roughly divided into two parts: A. inhibition and de-inhibition of carbon source and B. induction of methanol. But the detail of its mechanism is still remaining unclear.

Molecular cloning

First of all, we need to amplificated all the commercially synthesized plasmid to acquire enough amount for further study. After transformation, colony PCR is applied for confirmation. Then we go for plasmid extraction.

Fig1. Colony PCR confirmation of successful E.coli transfection.

Bright bands of identical sizes from colony PCR result demonstrates that 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.

Fig2. Colony PCR result of yeast after electroporation through electrophoresis.

The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.

SDS-PAGE

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.

Fig3. SDS-PAGE result of Laccase GS115 4CL LOX2 ACC pepACS DsbC+pepACS detecetion in the supernatant.

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.