The internal NdeI site that broke the lipase clone
An MSc student has amplified the 1,842 bp open reading frame of a bacterial lipase by PCR with primers designed to add NdeI and XhoI sites, intending to clone it into pET-28a(+) cut with the same enzymes so that the protein carries an N-terminal His6 tag. Ligation, transformation into BL21(DE3) and selection on kanamycin give 60 colonies. Colony PCR with T7 promoter and terminator primers gives a product of about 1,450 bp in 41 colonies instead of the expected 2,050 bp. The remaining colonies give no product. Sequencing of one 1,450 bp product shows the insert begins at the expected ATG but stops abruptly; the student's sequence analysis shows an NdeI recognition site (CATATG) at codon 214 of the native gene, which she had not checked. She proposes redoing the cloning using a partial NdeI digest, or alternatively switching to blunt-end cloning into the vector's SmaI site.
1. Evaluate the two proposed fixes (partial NdeI digestion and blunt-end cloning into SmaI) and recommend an alternative. Justify your choice in terms of reproducibility, screening effort and preservation of the N-terminal His6 tag.
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The 1,450 bp colony PCR product is the signature of internal cleavage: the NdeI site at codon 214 splits the 1,842 bp amplicon into roughly 640 bp and 1,200 bp fragments, and only a fragment with one NdeI end and one XhoI end can ligate productively into the vector, giving a truncated insert.
Partial digest. In principle the terminal NdeI site (created by the primer at the very end of a linear molecule) is cut faster than the internal site, so a limiting digest could enrich for single-cut molecules. In practice this requires careful titration of enzyme, units and time for every batch; the product is a mixture of uncut, terminally cut, internally cut and doubly cut molecules, only one of which is useful. It is poorly reproducible, low-yield, and would force restriction screening of many colonies. It is a fallback, not a first choice.
Blunt-end cloning into SmaI. Blunt ligation is inefficient, has no orientation control, needs a dephosphorylated vector and gives high background. More importantly, the NdeI site supplies the ATG that starts the fusion and fixes the reading frame relative to the His6 tag; moving the insert to a blunt site upstream or downstream means the reading frame and start codon must be re-created, usually by adding extra residues or by re-sequencing every clone.
Recommendation. Use overlap-extension (SOE) PCR or the equivalent to destroy the internal site silently: change CATATG to CACATG, which still encodes His-Met but is no longer an NdeI site. Then digest with NdeI/XhoI and clone as originally planned. Confirm success by digesting the new amplicon with NdeI (one linear 1,842 bp band instead of two fragments) and by full sequencing of the ORF. A ligation-independent method (Gibson/SLIC) is a valid alternative because it ignores internal sites entirely.
2. Once a clone is obtained, how would you prove that the His6 tag is in frame and that the full-length protein is being made? State the evidence you would accept and the results that would make you reject the clone.
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Sequence-based and protein-based evidence are both required.
Sequence. Sanger sequencing with the T7 promoter primer across the tag–insert junction, translated in silico: the six His codons and the thrombin site must be followed in frame by the insert's codons with no premature stop, and the predicted product must be 614 residues plus the tag (about 68–70 kDa). A frameshift at the junction is the commonest silent failure and cannot be excluded by restriction mapping or colony PCR size alone.
Expression. Grow the clone and the empty-vector control in BL21(DE3) to OD600 ~0.6, split, induce one flask with 0.5 mM IPTG (37 °C, 3 h) and keep the other uninduced. Run 12% SDS-PAGE of whole-cell lysates: accept the clone only if a band of the predicted size appears in the induced lane, is absent or much weaker in the uninduced lane, and is absent from the induced empty-vector control (which should instead show only the small tag peptide).
Identity of the band. Western blot with anti-His antibody must detect a single band at exactly that size. Ni-NTA purification giving the same band on a Coomassie gel is supporting evidence.
Reject the clone if the anti-His band runs at ~40 kDa or is absent after induction, if there are multiple anti-His bands (truncation or proteolysis), or if sequencing shows the junction out of frame. Mass spectrometry of the purified protein is the final confirmation if the N-terminal sequence matters.