Fig. 1. Cartoon representation of the vector construct. The DLD gene insert is located after the coding sequences of the ompA periplasmic targeting peptide, the Strep-tag and the Factor Xa cleavage site. Precise insertion of the human DLD gene was achieved by the application of the BsaI endonuclease. The vector is driven by the tet promoter while the encoded resistance is for Chloramphenicol (CamR). The advantages of the above mentioned genetic elements for the periplasmic expression of the human LADH protein are discussed in details in the text.

Fig. 2. (A) SDS–PAGE analysis of Strep-LADH expression and purification steps. Lanes: (1) MW standard, (2) non-induced bacteria, (3) bacteria after 24 h induction, (4) spheroplasts, (5) periplasmic content, (6) flow-thru after affinity chromatography, (7) flow-thru after re-administration of material represented in lane 6 onto affinity column, (8) flow-thru after column wash, (9) no sample, (10) eluted Strep-LADH, (B) Western blot identification of Strep-LADH through the tag by Strep-Tactin conjugated to HRP during expression and purification steps; some unspecific bindings of biotinylated proteins are seen before purification. Lanes: (1) MW standard, (2) non-induced bacteria, (3) bacteria after 3 h induction, (4) spheroplasts, (5) periplasmic content, (6) flow-thru after affinity chromatography, (7) flow-thru after column wash, (8) eluted Strep-LADH, (9) no sample (10) MW standard, (C) Western blot identification of Strep-LADH by monoclonal antibody against human LADH during expression steps. Lanes: (1) non-induced bacteria, (2) bacteria after 3 h induction, (3) bacteria after 9 h induction, (4) bacteria after 24 h induction, (5) spheroplasts, (6) no sample, (7) periplasmic content, (8) bacterial culture supernatant after 24 h induction; MW markers are not shown as they did not cross-react with the applied antibodies.

Fig. 3. (A) CD spectra of Strep-LADH and porcine LADH. The CD signatures of the two protein products from different origins are almost identical; the sequence homology between the two proteins is beyond 95%. The result represents correct folding and together with the result for specific activity of the enzyme product it suggests that the purified protein is fully functional. The MALDI-TOF MS analysis of the porcine heart LADH preparation used in our studies showed over 99% purity and correct MW (data not shown). The crystal structure of the LADH protein from human origin (PDB ID: 1ZMC) shows 34% helical and 26% β-sheet secondary structure arrangements; the CD signature shown here is in concert with the findings of the X-ray analysis. (B) Activity measurement of Strep-LADH in the reverse reaction. NADH consumption at 340 nm is detected upon addition of the enzyme in two concentrations (the second administration is with four times higher enzyme amount for clearly representing concentration dependence of activity). The specific activity of the enzyme is calculated to be 0.336 ± 0.014 μmol min⁻¹ mg⁻¹ that is 1.5 times higher than what we measured for the porcine LADH purchased from Sigma (0.220 ± 0.010 μmol min⁻¹ mg⁻¹) and it remains unchanged after Factor Xa cleavage (data not shown). Conditions: 50 mM K–PO₄ pH 7.3, 165 μM NADH, 1 mM lipoic acid, 37 °C.

Fig. 4. (A) SDS–PAGE detection of time-dependent Factor Xa cleavage of Strep-LADH. Lanes: (1) MW standard, (2) uncleaved Strep-LADH, (3, 4, 5 and 6) Strep-LADH after 30 min, 1 h, 2 h and 4 h cleavage by Factor Xa, respectively, (7) tag-cleaved LADH after elimination of Factor Xa by Xarrest Agarose. Factor Xa shows three, instead of two (light (15.7 kDa) and heavy (28.7 kDa) chains), bands after reductive SDS–PAGE since it is not completely activated (information from manufacturer), leaving a 9.5 kDa activation sequence on one population (50%) of the heavy chains. (B) Chromogenic detection through HRP-conjugated Strep-Tactin in Western blot analysis of time-dependent Factor Xa cleavage of Strep-LADH. Lanes: (1) MW standard, (2, 3, 4 and 5) Strep-LADH after 30 min, 1 h, 2 h and 4 h cleavage by Factor Xa, respectively, (6) non-cleaved Strep-LADH (placed to prove blotting efficiency). As seen, Factor Xa cleavage of LADH could not be detected as expected from sequence analysis. Uneven shading of membrane is the result of differences in membrane discoloration effects caused by the chromogenic reaction.

Fig. 5. (A) Analysis of Strep-LADH by in-gel digestion and tandem MS: A 19% sequence coverage (which is a high-standard identification for a protein) of the mitochondrial precursor human LADH protein by peptides identified in searches of the MS/MS data against the NCBI database was detected (shaded; see Table 1). Those peptides that have not reached high criteria in searches against the NCBI database, but have been identified against the human LADH protein alone (see Table 1 in italics) are underlined (3.1% additional sequence coverage). (B) MALDI-TOF MS analysis of LADH. As seen, the untagged protein product is intact, very clean (also confirming the SDS–PAGE data) and shows the theoretically expected MW for the protein (101,862.2 Da (with two FAD prosthetic groups); the experimental error is 0.14% that is in absolute terms 141.54 Da (approximately the MW of one amino acid)). The non-covalent homo-dimer partially dissociates in the MALDI source and that is why the signal for doubly charged molecular species is of similar intensity to the molecular ion. The signal-to-noise ratio is rather low in the spectrum due to the higher buffer/salt content of the protein sample that is handled by our MALDI on-plate washing protocol [45].

Supplementary data. 

Tandem MS analysis of in-gel digestion of Strep-LADH

Note. Data represented in this table are the results of a search of MS/MS data against the non-redundant NCBI database for the Strep-LADH protein. For the interpretation and acceptance thresholds of XCorr and DeltCN values, see the Materials and methods section. (*) and (#) after amino acids in the peptide sequences represent alkylation (+57) and oxidation (+16), respectively, caused by applied chemistry during analysis. Peptides in italics represent additional peptide hits arisen when MS/MS data were searched against the Strep-LADH sequence alone; DeltCN values cannot be interpreted for these hits (see Materials and methods).