2. Expression and Purification of Par-
2.4. Preparation of Par-4 expression vectors
Table 2.2 contains a list of all prepared Par-4 expression vectors. It lists the PCR primer sequences and the templates used for the PCR reactions. The expression vector and restriction
enzymes used for cloning are also given. PCR with Pwo or KOD Polymerase was performed
according to the manufacturers instructions. The PCR cycles for the two used polymerases were:
Pwo Polymerase KOD Polymerase
1.) 5 min at 94 ºC 1.) 2 min at 95 ºC
2.) 60 s at 94 ºC 2.) 20 s at 95 ºC
3.) 45 s at 60 ºC 3.) 15 s at 60 ºC
4.) 75 s at 72 ºC 4.) 45 s at 72 ºC
5.) 5 min at 72 ºC 5.) 5 min at 72 ºC
steps 2.) to 4.) are repeated 30 times steps 2.) to 4.) are repeated 30 times
The expression vectors and PCR products were digested with the corresponding restriction enzymes (Table 2.2) according to the manufacturers instruction. Cut vectors and inserts were ligated with T4-DNA Ligase according to the manufacturers instruction. The ligation reaction
was used to transform TOP10 or DH5α E. coli cells (Chapter 2.1.1.). Correct insertion of the
PCR product was assessed by sequencing of the plasmid (Allan Wilson Centre for Molecular Ecology and Evolution, Massey University, New Zealand). Plasmids with the correct sequence were used to transform the expression hosts E. coli BL21(DE3) CodonPlus or Rosetta(DE3). pET32TEV is an in-house expression vector created by Dr. Rose Brown (Institute of Molecular Biosciences, Massey University, New Zealand) and is derived from pET32a. The hexa-histidine tag and TEV cleavage site from pProEX-HTb was PCR amplified using the Primers 5’-GGTTCTGGCCATATGTCGTACTACCATCACCATCA-3’ (Fw) and 5’-TGCCAAG CTTTCTAGAGGATCCGGATTGAAAATACAGGTTTTCGGTC-3’ (Rv). The PCR product was cut with Msc I and BamH I and then used to replace the original sequence, with a Thrombin and Enterokinase cleavage site, in pET32a. The vector name HpMal-c2P is used instead of HMBP-3C [187] to avoid any confusion with the hexa-histidine and MBP-tagged 3C protease (HMBP-3Cpro).
Construct Name Forward Primer (5’-3’) Reverse Primer (5’-3’) Template Restriction Enzymes Expression Vector Par-4(1-332)WT Par-4(1-332)WT Par-4(1-332)G40G Par-4(1-332)G40G Par-4(1-290)G40G Par-4(286-332)WT Par-4(286-332)WT Par-4(286-332)WT Par-4(286-332)N313I CAGGAATTCCATGGC GACCGGCGGCTATCG GAGC CAGGTCGACTTACC TTGTCAGCTGCCCA ACAAC
rat Par-4 cDNA EcoR I / Sal I pGex-4T-3
CAGGAATTCAAATGG CGACCGGCGGCTATC GGAG CAGGTCGACTTACC TTGTCAGCTGCCCA ACAAC
rat Par-4 cDNA EcoR I / Sal I pProEX-HTb
CAGGGATCCATGGCG ACCGGCGGCTATCGG AG CTTGGCGGCTGGAT CTCCGCCGCTCGAA C
rat Par-4 cDNA BamH I pET32a
GTTCGAGCGGCGGAG ATCCAGCCGCCAAG
CAGGTCGACTTACC TTGTCAGCTGCCCA ACAAC
rat Par-4 cDNA Sal I
CAGGGATCCATGGCG ACCGGCGGCTATCGG AG CAGGTCGACTTACC TTGTCAGCTGCCCA ACAAC Par-4(1-332)G40G
in pET32a BamH I / Sal I pET32TEV
CAGGGATCCATGGCG ACCGGCGGCTATCGG AG CCGGAAGCTTTTAT TCTTCTTTATCTTG CATCAG Par-4(1-332)G40G
in pET32a BamH I / Hind III pCFE-TrxH-TEV
GCACTAGGATCCCAA GATAAAGAAGAAATG
CAGGTCGACTTACC TTGTCAGCTGCCCA ACAAC
rat Par-4 cDNA BamH I / Sal I pCFE-GST-3C
GCACTAGGATCCCAA GATAAAGAAGAAATG
CAGGTCGACTTACC TTGTCAGCTGCCCA ACAAC
rat Par-4 cDNA BamH I / Sal I pCFE-GST-TEV
GCACTAGGATCCCAA GATAAAGAAGAAATG
CAGGTCGACTTACC TTGTCAGCTGCCCA ACAAC
rat Par-4 cDNA BamH I / Sal I pGex-6P-3
GCACTAGGATCCCAA GATAAAGAAGAAATG
CCTGCTTTAGTTGC TCGATTTCGTC
Par-4(286-332)
WT in pGex-6P-3 BamH I pGex-6P-3
GACGAAATCGAGCAA CTAAAGCAGGA CAGGTCGACTTACC TTGTCAGCTGCCCA ACAAC Par-4(286-332) WT in pGex-6P-3 Sal I
Table 2.2− Preparation of Par-4 expression vectors. Primer sequences and DNA templates for PCR amplification are given. Restriction enzymes and host vectors are also listed.
2.4.2. Results and Discussion
The Par-4 constructs made for this study were designed so that they produce proteins, which are of biological significance (Chapter 1.2.). All sequence numberings refer to the rat isoform of Par-4. Note that the cDNA of rat Par-4 was used as a template for PCR reactions during the preparation of Par-4 expression vectors. Full-length constructs of Par-4 (Par-4(1-332)) were made as reference constructs to all other constructs comprising deletion mutants and single domains of Par-4. To take advantage of an N-terminal BamH I site, an internal BamH I site present in the rat Par-4 cDNA was removed by PCR (Table 2.2). Constructs with this silent point mutation are denoted G40G instead of WT (wild-type). Using a BamH I site at the N-terminus reduces the N-terminal cloning artefact for most expression vectors, especially for vectors of the pET series. Also transfer between different expression vectors is simplified as most BamH I sites
are in the same translation frame in various vectors. As mentioned in the Introduction, ectopic over-expression of Par-4 in PC3 or melanoma cells showed that the LZ domain is essential for the sensitivity to apoptotic stimuli [55]. Deletion mutants that lack the LZ domain, however, did not confer enhanced sensitivity [55]. Two such LZ deletion mutants were made (Par-4(1-265) and Par-4(1-290)). The Par-4(1-290) constructs contain 15 additional residues of the C-terminal CC region (residues 254-332) [46]. The rationale was to determine if coiled coil formation is possible without the LZ domain. The SAC domain is sufficient for apoptosis induction in cancer cells [50]. Therefore, a similar construct as used in this previous study comprising only the SAC domain was made (Par-4(137-195)). A construct comprising the CC domain (Par-4(240-332)) was made as it was shown that this domain is able to act in a dominant negative manner abrogating the pro-apoptotic function of Par-4 [55]. Various constructs of the LZ domain (Par-4 (286-332)) were made as these constructs show an interesting pH dependent folding behaviour [184]. However, a physiological function has not been described for this construct. The Par-4 (286-332)W285 mutant was created to determine the protein concentration by A280 measurements as the wild-type LZ domain does not contain aromatic residues. The rationale for
the creation of the Par-4(286-332)D305K and E310K point mutants is described in Dutta et. al.
[185]. In short, these two mutants adopt a coiled coil conformation independent of pH.
Sequencing confirmed correct insertion of the PCR products into the vectors and verified the right nucleotide sequence for almost all constructs. Only one Par-4 expression vectors listed in Table 2.2 could not be created during the PhD (see below). Some of the vectors used in this study were created by other researchers in a similar way and their acknowledgements are given below. The HpMal-c2P expression vectors of Par-4(286-332)WT, Par-4(286-332)W285, Par-4 (286-332)D305K and Par-4(286-332)E310K [184,185], and the pGex-6P-3 expression vector of Par-4(240-332)WT were kindly provided by Dr. Kaushik Dutta (New York Structural Biology Center, New York, USA). The HpMal-c2P expression vectors of Par-4(1-332)WT, Par-4(1-265) WT and Par-4(1-290)WT were prepared by Dr. Komala Ponniah (Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand). Par-4(137-195)WT in pET32TEV was created by Sachin Kate (Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand).
The only Par-4 expression vector that could not be created during the PhD was Par-4(1-332) WT in pGex-4T-3. Even though a similar vector is described in the literature [53], all attempts to create this vector resulted in recombination. Sequencing of several clones after transformation showed that both the pGex-4T-3 vector and the PCR product were cut properly at the 5’ and 3’
end and that the PCR product and the vector ligated properly. However, in all sequenced constructs the central part of Par-4, approximately residues Ser 8 to Asp 308, was lost by recombination. Restriction digest, ligation and transformation were repeated, however, gave
similar results. Additionally, different E. coli strains such as DH5α, TOP10 or BL21(DE3)
CodonPlus were tested, but all gave similar results. As expression results of Par-4(1-332)G40G from pET32a were promising, preparation of the pGex-4T-3 vector was abandoned. Re-cloning of this construct was not tried as Par-4(1-332) can be purified in high purity from a different construct.
Due to improper cleavage of the 5’-end of the PCR insert for pCFE-GST-3C four additional nucleotides were included between the ribosome binding site and the initiation codon. In case of Par-4(286-332)WT in pCFE-GST-3C (Chapter 2.11.2.) this increased spacing between the ribosome binding site and the initiation codon still allowed expression. However, the protein expression levels were comparable to the pGex-6P-3 (Chapter 2.11.3.) vector suggesting that the increased spacing decreased the expression efficiency. pCFE-GST-3C has been re-cloned to remove these four additional residues, but it has not been tested for expression with new inserts.