Construction of the Recombinant P pastoris Expression Vectors

The virus gene sequences were first inserted into the appropriate yeast transfer vector. In these vectors, the expression of the recombinant protein is under the control of the alcohol oxidase (AOX 1) promoter (11,12). The AOX 1 promoter is inducible by the presence of methanol in the absence of any other carbon source. As P. pastoris is a methylotrophic yeast, it consumes methanol readily, thus generating large quantities of the alcohol oxidase to as much as >30% of the total soluble protein. In this way, high-level expression of the genes of interest could be driven from the AOX 1 promoter. In addition, these vectors also contain selectable markers, different types of affinity tags for easy protein purification, and also the use of secretion signals to target the recombinant protein into the growth medium.

The DEN E glycoproteins were constructed under the AOX 1 promoter in two expression vectors, pHIL-S1 and pHIL-D2 (Invitrogen). Both vectors carry the HIS4 gene for selection in his4 strains, and the 3' AOX 1 sequences for integration into the host genome. In addition, the pHIL-S1 vector contains the P. pastoris alkaline phosphatase signal sequence, which targets transport of the recombinant protein into the yeast secretory pathway. The methods used for the expression of recombinant proteins from P. pastoris transformed with pHIL-S1 and pHIL-D2 are described under Subheadings 3.1.1.-3.1.3.

3.1.1. Construction of DEN E Gene in pHILSI Expression Vectors

1. Amplify the GST coding sequence with GST-specific forward primer CRGSTX and reverse primer CLANKS, from vector pGEX-KG with PCR.

2. Analyze and elute the 0.7-kbp PCR product from 1.2 % agarose gel electrophore-sis using standard methods.

3. Digest the purified product with XhoI and StuI, and ligate into the same sites in pHIL-S1 to generate pHIL-S1/GST (see Note 1).

4. Design specific primers to clone the E gene of different lengths (1 to 495 amino acids, 1 to 401 amino acids, and 1 to 213 amino acids), and engineer restriction enzyme sites into the 5' end of the sequences.

5. PCR amplify the full-length of E gene (1 to 495 amino acids) with primers DIR899E and DIFYE, using plasmid pAD97 as the template.

6. PCR amplify truncated E genes (1 to 410 amino acids, and 1 to 213 amino acids) with primers CRGSTX and DIFE401A, and CRGSTX and DIF2X00X respectively, using vector pGEX-KG/EX20 as the template.

7. Analyze all PCR products in a 1% agarose gel electrophoresis and size-fractionate the PCR products of interest from the gel.

8. Sequence all PCR product completely with dideoxy sequencing method using Sequenase Version 2.0.

9. Perform EcoRI digestion on the purified PCR product containing the full E gene (1 to 495 amino acids), and ligate to the EcoRI-digested pHIL-S1/GST.

10. Perform XhoI/StuI digestion on the purified PCR product obtained for the truncated E gene (1 to 401 amino acids), and ligate to the same sites of pHIL-S1.

11. Digest the PCR product obtained for the truncated E gene (1 to 213 amino acid) with XbaI, blunt-ended with T4 polymerase, and ligated to XhoI/SmaI digested pHIL-S1.

12. Linearise the recombinant vectors with BglII digestion and proceed with the transformation into spheroplasts (see Subheading 3.2.1.).

3.1.2. Construction of DEN E Gene in pHIL-D2 Expression Vectors

1. Design DEN 1 specific primers covering the genes CprME, and engineer EcoRI sites into the 5' end of the sequences.

2. PCR amplify CprME with primers DIR81E and DIFYE, using plasmid pFA/1 as the template.

3. Analyze the PCR product in a 1% agarose gel electrophoresis.

4. Size-fractionate the 2.3 kbp PCR product of interest from the agarose gel.

5. Sequence the PCR product completely using the dideoxy sequencing method to confirm the sequence.

6. Perform EcoRI digestion on the purified PCR product, as well as the P. pastoris expression vector pHIL-D2.

7. Ligate the EcoRI digested PCR product to the same sites on pHIL-D2.

8. Linearise the recombinant vector, pHIL-D2/CprME, with NotI digestion and proceed with the transformation into spheroplasts (see Subheading 3.2.1.).

Figure 1 is a schematic diagram depicting the construction of expression vectors. DEN E proteins of different length were constructed under the AOX1 promoter in the two expression vectors. Different lengths of the E gene, fused at its 5' end with a GST tag for affinity purification later, were expressed in P. pastoris using pHIL-S1 (Fig. 1A). Full-length E proteins (GST E495, representing 1-495 amino acids) and truncated forms (GST E213, and GST E 401 representing 1-213 and 1-401 amino acids respectively from the N-ter-minal) were successfully expressed. Using a second vector, pHIL-D2, which is designed for intracellular protein expression but containing the same AOX1 promoter, the E protein was co-expressed as part of the CprME construct (Fig. 1B). In the latter case, the DEN virus glycoproteins are targeted into the yeast secretory pathway by the endogenous virus signal sequences which are present in the virus glycoproteins.

Fig. 1. Construction of the Pichia pastoris transfer vectors containing the DEN virus protein sequences. (A) PCR products containing the glutathiones-transferase (GST) and regions of the E protein sequence were inserted into pHIL-S1. The GST (hatched) and E protein (clear) coding regions and E protein transmembrane regions (black) are highlighted. S represents the signal sequence. (B) The DEN virus sequence containing the C, PrM, and E protein were inserted into pHIL-D2. In all cases the protein expression was under the control of the AOX1 promoter.

Fig. 1. Construction of the Pichia pastoris transfer vectors containing the DEN virus protein sequences. (A) PCR products containing the glutathiones-transferase (GST) and regions of the E protein sequence were inserted into pHIL-S1. The GST (hatched) and E protein (clear) coding regions and E protein transmembrane regions (black) are highlighted. S represents the signal sequence. (B) The DEN virus sequence containing the C, PrM, and E protein were inserted into pHIL-D2. In all cases the protein expression was under the control of the AOX1 promoter.

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