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The S. pneumoniae pbps were cloned into the expression vector pET46 Ek/LIC (vector map displayed in Figure 2.1). The vector encodes a hexa-histidine tag, which yields an N-terminal fusion upon protein translation, a desirable property for future protein purification considerations. The N-terminal fusion tag should not interfere with the enzyme activity, being located far from the catalytic sites.

3.3.2.1. Primer design with an engineered dodeca-histidine fusion tag

The primers for the PCR were designed according to the Ek/LIC cloning system, following the manufacturer’s instructions (Section 2.3.8, Figure 2.2). A sequence of nucleotides specific to the cloning procedure was incorporated 5’ and 3’ to the insert specific sequences of the sense and antisense primers respectively. An Nde I site and codons for six histidine residues were additionally integrated into the sense primer directly 5’ to the insert specific sequence (Table 3.4). Upon expression of the fusion protein, the vector and primer-encoded six histidine residues would be in close proximity (separated by a short sequence of amino acid residues), essentially creating a dodeca-histidine tag (proposed to aid protein purification) (Figure 3.1). The Nde I restriction site was incorporated as a precaution, in case an alternative cloning method was required. The start codon, ATG, was eliminated from the gene-specific sequence, due to its presence in the Nde I site. The stop codon, TAA, remained in the gene-specific sequence of the antisense primer to terminate translation.

3.3.2.2. Sequences of primers for the pET46 Ek/LIC cloning

The sense and antisense primers for the ligation independent cloning (LIC) of S. pneumoniae PBP variants into the pET46 vector are recorded in Table 3.4. It is noteworthy that the full-length S. pneumoniae 5204 pbp2b and pbp2x gene sequences are unknown; the sequences at the 5’ ends (and thus N-termini of the proteins) were undefined. The 3’ sequences of S. pneumoniae 5204 pbp1a, pbp2b and pbp2x are known, thus the insert specific sequences of the antisense primers were designed accordingly. The S. pneumoniae 5204 pbp1a gene sequence directly at the 5’ end does not differ from that of the D39 strain, permitting identical sense primers to be employed. It was therefore assumed that S. pneumoniae 5204 pbp2b and pbp2x gene sequences at the 5’ ends also do not differ from those of the D39 strain, allowing sense primers to be designed (identical to the sense primers of the strain D39 PBPs). The 5’ ends of the gene sequences from the penicillin-resistant strain are unlikely to deviate from the penicillin-sensitive strain as they encode residues located distant to the transpeptidase domain, which is subject to antibiotic selection pressures. Any disregarded amino acid changes at the N-terminal would be unlikely to affect the resistance mechanism of the S. pneumoniae 5204 PBP or impede its activity.

S. pneumoniae

pbp gene target

Description of

primer Primer sequence (5’-3’ orientation)

Sense primer (FL) GAC GAC GAC AAGATG CAT ATG CAC CAT CAC CAT CAC CAT AAC AAA CCA ACG ATT CTG CGC C

Sense primer

(-TM, !30) GAC GAC GAC AAG ATG CAT ATGCAC CAT CAC CAT CAC CAT GTT TTT TTC TAC TAC GTT AGC AAG

D39 pbp1a

Antisense primer GAG GAG AAG CCC GGT TTA TGG TTG TGC TGG TTG AGG ATT C

Sense primer (FL) GAC GAC GAC AAGATG CAT ATG CAC CAT CAC CAT CAC CAT AAC AAA CCA ACG ATT CTG CGC C

5204 pbp1a

Antisense primer GAG GAG AAG CCC GGT TTA TGG TTG TGC TGG TTG AGG ATT C

Sense primer (FL) GAC GAC GAC AAG_{AAC AGC} ATG CAT ATGCAC CAT CAC CAT CAC CAT AGA CTG ATT TGT ATG AGA AAA TTT

Sense primer

(-TM, !39)

GAC GAC GAC AAGATG CAT ATG CAC CAT CAC CAT CAC CAT CAG GTT TTG AAC AAG GAT TTT TAC

G

D39 pbp2b

Antisense primer GAG GAG AAG CCC GGT CTA ATT CAT TGG ATG GTA TTT TTG ATA CAG

Sense primer (FL) GAC GAC GAC AAG_{AAC AGC} ATG CAT ATGCAC CAT CAC CAT CAC CAT AGA CTG ATT TGT ATG AGA AAA TTT

5204 pbp2b

Antisense primer GAG GAG AAG CCC GGT CTA ATT CAT TGG ATG GTG TTG G

Sense primer (FL) GAC GAC GAC AAGATG CAT ATGCAC CAT CAC CAT CAC CAT AAG TGG ACA AAA AGA GTA ATC CG

Sense primer

(-TM, !48)

GAC GAC GAC AAGATG CAT ATGCAC CAT CAC CAT CAC CATGGG ACA GGC ACT CGC TTT GGA

ACA G

D39 pbp2x

Antisense primer GAG GAG AAG CCC GGT TTA GTC TCC TAA AGT TAA TGT AAT TTT TTT AAT G

Sense primer (FL) GAC GAC GAC AAGATG CAT ATGCAC CAT CAC CAT CAC CATAAG TGG ACA AAA AGA GTA ATC CG

5204 pbp2x

Antisense primer GAG GAG AAG CCC GGT TTA GTC TCC TAA AGT TAA TTT AAT TTT TTT AAT G

Table 3.4: The primers required to amplify different variants of pbp1a, pbp2b and pbp2x for cloning into the pET46 vector. The regions in bold, black type indicate the nucleotides that are a cloning-specific requirement as dictated in the manufacturer’s instructions. The sequence GAC GAC GAC AAG ATG encodes the enterokinase cleavage site that is incorporated at the 5’ terminal of the gene sequence. The bold, red type represents the Nde I restriction enzyme cleavage site. The bold, blue type shows the engineered codons for the extra six histidine residues. The plain text shows the 5’ or 3’ gene specific sequences, essential for annealing during the PCR. FL, full-length sequence; -TM, sequence devoid of the transmembrane and cytoplasmic region, where the length of protein truncation is denoted.

The ATG codon, required for the functionality of the protein enterokinase restriction site, and the DNA Nde I site, were assumed not to act as start codons; a vector- specific ATG codon, locating 5’ to the vector-encoded histidine tag, fulfilled this role, being in close proximity to the vector ribosomal binding site. Following protein expression, a recombinant protein would be produced as illustrated in Figure 3.1.

Figure 3.1: Domain organisation of the recombinant proteins. The recombinant protein contains an N-terminal vector-encoded cleavable six histidine tag (6His1), separated from a primer-encoded non-cleavable six histidine tag (6His2) (not anticipated to interfere with enzyme activity) by a short sequence of amino acids encompassing an enterokinase cleavage site (Ek) and the amino acids encoded by the Nde I site (histidine and methionine). The locations of the start (ATG) and stop (TAA) codons are indicated. N, amino-terminus; C, carboxy-terminus.