Plasmid Vector Design

3.6.1 Cloning into the Plasmid Vector pET28a for Antigen Production

In order to obtain proteins of the PIGs to be studied for antibody production, these proteins were over-expressed using a suitable plasmid vector. The vector chosen was pET28a⁺ (see chapter 3.2.3), which codes for a histidine tag, simplifying the isolation of the fusion proteins.

Starting with full-length cDNA clones, cDNA was amplified by PCR using specially designed primers (see Table 3-16), in order to affix an additional restriction site to the 5’-end and specially designed primers in order to affix an additional restriction site to the 3’-end or a λ-phage-specific primer at the 3’-end of the clone. The 5’-primers were also designed to exclude the potential secretion signals from the newly produced DNA to be cloned into the vector and to cause an in-frame insertion in the plasmid construct. The λ-phage-specific 3’-primers were chosen to include the cloning site (NotI, EcoRI) from the phage DNA. By including this cloning site or using the affixed restriction site at the 3’-end, the PCR product can be digested with the corresponding restriction enzymes as listed in the following table 3-27:

Table 3-28 List of Restriction Enzymes Used for Cloning into pET28a⁺ PIG cDNA/

Name of Construct

Restriction 5’-end

Manufacturer Restriction 3’-end

Manufacturer

PIG5 NdeI NEB, Frankfurt/M, D NotI MBI, St.Leon-Rot, D

PIG5n SacI Stratagene,

Amsterdam, NL

BamHI Stratagene, Amsterdam, NL PIG7 NdeI NEB, Frankfurt/M, D EcoRI Stratagene, Amsterdam, NL PIG9 NdeI NEB, Frankfurt/M, D EcoRI Stratagene, Amsterdam, NL

PIG9s SacI Stratagene,

Amsterdam, NL

BamHI Stratagene, Amsterdam, NL

PIG9l SacI Stratagene,

Amsterdam, NL

BamHI Stratagene, Amsterdam, NL PIG14 NdeI NEB, Frankfurt/M, D EcoRI Stratagene, Amsterdam, NL PIG15 NdeI NEB, Frankfurt/M, D EcoRI Stratagene, Amsterdam, NL

PIG23 SacI Stratagene,

Amsterdam, NL

XhoI Boehringer, Mannheim, D

The plasmid vector pET28a⁺ and the corresponding inserts were digested with the appropriate restriction enzymes according to the manufacturer’s manual in order to generate compatible ends for cloning. Afterwards the enzymes were heat-inactivated for 15 min at 65°C. To remove the phosphate groups from the 5’-ends, the vector was dephoshorylated with shrimp alkaline phosphatase (USB™, Bad Homburg, D). This served to prevent self-ligation of insufficiently digested vector.

After the various above mentioned process steps, the inserts and vector were ligated. Ligation was performed according to Maniatis et al. (1982) using 10 ng plasmid, 30 ng insert DNA and 1 unit T4 ligase in Ligase buffer (USB™, Bad Homburg, D). The ligation mix was incubated over night in ice-cold water in a Styrofoam box. The resulting newly generated plasmid was transformed into E. coli TG1, multiplied and the modified plasmid vector isolated (see chapter 3.8.2.2).

The resulting modified vector pET28a:insert (see Figure 3-1) codes for the start codon and a histidine tag originating from the vector DNA, part of the PIG cDNA with the stop codon, some non-coding sequence and part of the phage DNA, including the restriction site.

pET28a

Figure 3-1 Model of Plasmid Vector pET28a⁺ with Insert for Antigen Production

3.6.2 Cloning into Yeast Shuttle Vector pDR195 for Heterologue Protein Production The yeast shuttle vector pDR195 (see chapter 3.2.3) can be multiplied in E. coli and then be used to transform yeast cells. Because pDR195 offers only a few restriction sites in its cloning site, only one restriction site (NotI) was used to clone the insert into the yeast shuttle vector.

The plasmid Vector pDR195 and the corresponding inserts of the studied PIGs (PIG5, PIG7, PIG14, PIG15) were digested with the appropriate restriction enzyme NotI according to the manufacturer’s manual (MBI Fermentas GmbH, St. Leon-Rot, D) in order to generate compatible ends for the cloning. In contrast to the pET28a cloning process, the inserts for cloning into pDR195 include the putative signal sequences of the studied PIGs. To remove the phosphate groups from the 5’-ends, the vector was treated with shrimp alkaline phosphatase (USB™, Bad Homburg, D). This served to prevent self-ligation of the vector. Afterwards the enzymes were heat inactivated for 15 min at 65°C.

After the various above mentioned process steps, the inserts and vector were ligated as described in 3.6.1. The resulting plasmid was transformed into E. coli TG1 and multiplied.

After the right orientation of the insert was controlled by PCR, the plasmid was isolated and used to transform yeast cells (see chapter 3.7.2).

3.7 Transformation

3.7.1 Transformation of Bacterial Cells

The transformation of bacterial cells served either to multiply the plasmid vectors or to facilitate further over-expression of the fusion protein. Transformation was performed according to Mandel and Higa (1970). First, E. coli strains were grown in LB medium for about 3 h at 37°C and 275 rpm until an OD600 of 0.5 - 0.6 was reached. 5 ml aliquots of this culture were then put on ice before being centrifuged for 10 min at 4°C and 1’000 g. The supernatants were discarded, and each pellet was resuspended in 5 ml ice-cold 0.1 M CaCl2

solution and put on ice for 30 min. The centrifuging step was repeated and the supernatants discarded. Each pellet was resuspended in 500 µl ice-cold 0.1 M CaCl2 solution and put on ice for between 1 h and 18 h. In each case, 100 µl of the suspension were mixed with plasmid- or ligation make-up and incubated on ice for 30 min. The subsequent heat shock (90 sec, 42°C) was followed by another five minutes on ice. 900 µl LB medium were added to the transformation mixture and incubated for one hour at 37°C. Afterwards 100 µl respectively 300 µl of the mixture were plated on LB plates containing the required antibiotic. The plates

His-tag

START Part of the cDNA incl. STOP Part of λ phage

were incubated at 37°C for 12 h to maximum 24 h, until bacterial colonies became visible. To verify whether the desired plasmid/insert construction was present, single colonies were then picked and controlled by PCR.

3.7.2 Transformation of Yeast Cells

Yeast transformation was used to over-express proteins in yeast, and was performed according to Elble (1992). Five to six colonies of the Saccharomyces cerevisiae strain SEY2102 (Emr et al. 1983) (grown on SC-maltose + uracil plate) were mixed with the pDR195 constructs before being mixed with 0.5 ml of PLATE-solution (see Table 3-29). The transformation make-up was incubated over night at room temperature (RT) before being subjected to heat shock (15 min, 42°C). The mixture was then briefly spun down and the pellet resuspended in 250 µl H2O. 100 µl of each transformation make-up were plated on SC-Maltose-agar (without uracil) and incubated for about six days at 30°C, until yeast colonies became visible. To verify whether the desired plasmid/insert construction was present, single colonies were then picked and controlled by PCR.

Table 3-29 PLATE-Solution (Elble 1992)

Contents Concentration

PEG 3350 40 %

Lithiumacetat 100 mM

Tris-HCl, pH 7.5 10 mM

EDTA 1 mM