Part:BBa_K2924042

AOX promoter + Lactoferrin + HisTag + AOX Terminator

The heterologous expression of proteins has an important industrial role, e.g. for the production of insulin¹. Identification of new peptide and protein pharmaceuticals and the optimization of the expression of known pharmaceuticals represent a huge research sector. Around 155 pharmaceuticals and vaccines developed by bio-pharmaceutical companies were approved by the US Food and Drug Administration in 2002, the amount of approved recombinant proteins quickly rose to over 200 in 2009². Besides insulin, medically important proteins like albumin and the human growth hormone (HGH) are produced by microbes or higher organisms. Small proteins are usually expressed in prokaryotic organisms like Escherichia coli, which enables easy, quick and cheap protein expression³. Disadvantages are the lack of post-translational modification and glycosylation, the difficult expression of large proteins and proteins with disulphide bonds^3,4. These drawbacks can be compensated by using eukaryotic yeast as an expression host. Two of the primarily used yeast expression systems are Saccharomyces cerevisiae and Pichia pastoris ⁵.

Fig. 1: The genetic organisation of the P. pastoris expression system with the AOX Promoter BBa_K2924039 followed by the target gene and C-terminally fused to a His-tag and AOX1 Transcription Terminator BBa_K2924040. The vector is pPICZB. E: Eco72I. N: NotI

In P. pastoris, the AOX1 promoter is methanol inducible and therefore the AOX is highly expressed in the presence of methanol⁶. This leads to high recombinant protein yields of genes introduced into P. pastoris under influence of the AOX1 promoter⁷. The pPICZB Vector from the EasySelect™ Pichia Expression Kit from Thermofisher Scientific was used, which contains an inducible AOX1 promoter and a Zeocin™ resistance gene.

The lactoferrin coding sequence originates from the organism Bos Taurus and was synthesized commercially. In addition, a NotI and an Eco72I interface were placed at both ends of the gene to clone the gene into the pPICZB vector (Fig. 1).

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 1
  Illegal BamHI site found at 2232
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1220
  Illegal BsaI.rc site found at 2215
  

Characterization

Fig. 2: Relative gene expression level of Lactoferrin in P. pastoris 24 h after methanol induction. The expression level of each gene was normalised to the housekeeping genes ARG4 and TAF10. The highest expressing strain of each construct was set to 100 % to gain relative expression levels.

After transformation of the construct shown in Fig. 1 and subsequent expression of lactoferrin in our Pichia strain, we investigated whether an increase in lactoferrin gene expression could be observed by performing RT-qPCR. To measure the expression levels, P. pastoris RNA was isolated, cDNA was synthesised and as a negative control (RNA control), the same reaction was run without adding the reverse transcriptase. A negative control strain, containing only the empty vector, was also included. The qPCR was run with lactoferrin-specific qPCR primers. Data were normalized to the two Pichia housekeeping genes PpARG4 and PpTAF10. Fig. 2 shows the relative expression of lactoferrin. While the EVC (blue) shows no expression at all, lactoferrin expression (red) is high. While the RNA control also showed a signal, this is only minor and likely due to residual gDNA contamination. In summary, we were able to show high expression of lactoferrin in P. pastoris based on qRT-PCR. This is the first step towards establishing P. pastoris as one of our milk protein production chassis. While there was no time to check protein amount and activity, we are confident that this organism is an excellent addition to our repertoire.

References

1: Ebina Y, Edery M, Ellis L, Standring D, Beaudoin J, Roth RA, Rutter WJ (1985) Expression of a functional human insulin receptor from a cloned cDNA in Chinese hamster ovary cells. Proc Natl Acad Sci U S A 82: 8014–8018

2: Demain AL, Vaishnav P (2009) Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv 27: 297–306

3: Terpe K (2006) Overview of bacterial expression systems for heterologous protein production: From molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 72: 211–222

4: Tran A-M, Nguyen T-T, Nguyen C-T, Huynh-Thi X-M, Nguyen C-T, Trinh M-T, Tran L-T, Cartwright SP, Bill RM, Tran-Van H (2017) Pichia pastoris versus Saccharomyces cerevisiae: a case study on the recombinant production of human granulocyte- macrophage colony-stimulating factor. BMC Res Notes 10: 148

5: Byrne B (2015) Pichia pastoris as an expression host for membrane protein structural biology. Curr Opin Struct Biol 32: 9–17 Lin-Ceregh

6: Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24: 45–66

7: Byrne B (2015) Pichia pastoris as an expression host for membrane protein structural biology. Curr Opin Struct Biol 32: 9–17.

8: Lin-Cereghino J, Wong WW, Xiong S, Giang W, Luong LT, Vu J, Johnson SD, Lin- Cereghino GP (2005) Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. Biotechniques 38: 44–48