A lower isoelectric point increases signal sequence-mediated secretion of recombinant proteins through a bacterial ABC transporter

Abstract

Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. Pseudomonas fluorescens has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF in vivo when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown. Here, we recombined LARD3 with several proteins and examined their secretion through TliDEF. We found that the proteins secreted via LARD3 are highly negatively charged with highly-acidic isoelectric points (pI) lower than 5.5. Attaching oligo-aspartate to lower the pI of negatively-charged recombinant proteins improved their secretion, and attaching oligo-arginine to negatively-charged proteins blocked their secretion by LARD3. In addition, negatively supercharged green fluorescent protein (GFP) showed improved secretion, whereas positively supercharged GFP did not secrete. These results disclosed that proteins' acidic pI and net negative charge are major factors that determine their secretion through TliDEF. Homology modeling for TliDEF revealed that TliD dimer forms evolutionarily-conserved positively-charged clusters in its pore and substrate entrance site, which also partially explains the pI dependence of the TliDEF-dependent secretions. In conclusion, lowering the isoelectric point improved LARD3-mediated protein secretion, both widening the range of protein targets for efficient production via secretion and signifying an important aspect of ABC transporter-mediated secretions.

Keywords: ABC transporter; isoelectric point; membrane potential; membrane transport; protein secretion; protein translocation; recombinant protein expression.

Figure 1.

Secretion of selected proteins. A and B, Western blotting image showing the expression and secretion of the target proteins. The cell samples show the amount of the protein that remains in the cytoplasm, and the supernatant samples represent the amount of protein that is localized to the extracellular space. For comparison, equivalent amounts of cell extract and culture supernatant (16 μl) were loaded onto the gel and were analyzed via Western blotting. 50 ng of TliA was loaded in the middle of the gel as a reference. Two other Western blottings were obtained from different culture samples. All of the unpresented results exhibit similar patterns. Below the images, there are Western blottings of the same samples but with primary antibody against cytosolic Neo, the neomycin/kanamycin phosphotransferase 2 protein. The nonspecific lysis or leakage is minimal in all samples except capsid.

Figure 2.

Correlation between % secretion of the target proteins and their pI values. A, protein isoelectic point and secretion. The pI value of the target proteins is calculated from the sequence, including the attached LARD3, as provided in supplemental section A. The proteins that have not been secreted have their bars colored red. AP, EGF, and PLA1 are proven to be secreted in previous studies and are added in this figure. B, secretion percentage and pI. The percent secretion values were calculated by supernatant signal divided by the sum of supernatant and cell signals. Three different biological replicates (independent culture samples) of the experiment in Fig. 1 were used for the quantitative analysis. Two highly basic outlier proteins that were not secreted, MAP (pI = 9.61) and capsid (pI = 9.25), were excluded from the plot. There was a negative correlation between the protein pI and their % secretion.

Figure 3.

Secretion of lunasin and its derivatives. Lunasin and its derivatives with different lengths of oligo-aspartic acid tail were expressed and secreted via LARD3 attachment to determine the optimal length of the oligo-aspartic acid sequence in P. fluorescens expression and secretion system. A, expression of lunasin and its derivatives in the cell and supernatant was detected via Western blotting. 36-μl eq of cell extract and supernatant were loaded onto the gel and were analyzed via Western blotting. B, protein sequence and domain structure of lunasin and its derivatives whose length of the aspartic acid tail is modified. They were named as lunasin-D0, lunasin-D5, original lunasin (D9), lunasin-D15, and lunasin-D20.

Figure 4.

Structures of plasmids used. A, structure of the pDART plasmid near the MCS. tliD, tliE, tliF, and the LARD3-attached fusion protein are controlled in a single operon. MCS is directly followed by the LARD3 gene, and thus the inserted target gene is expressed with LARD3 attached on its C terminus. B, structure of pFD10 plasmid that attaches D10 sequence at the N terminus. The D10 gene directly follows the start codon and is located right before the MCS and LARD3. C, structure of the pBD10 plasmid, which attaches D10 sequence at the C-terminal side, but before LARD3. The D10 gene is located between the MCS and LARD3.

Figure 5.

Expression of TliA derivatives in pFD10 and pBD10. A, Western blot of NKC-TliA in pFD10 and pBD10. The Western blottings were performed in a manner identical to that of the previous figures. Secretion strongly improved in both pFD10 and pBD10 when compared with pDART. B, enzyme plate assay of NKC-TliA in different plasmids. C, Western blot of CTP-TliA in pFD10 and pBD10. Secretion strongly increased in pBD10. D, enzyme plate assay of CTP-TliA in different plasmids. pBD10 exhibits a major increase in secretion. Two other Western blot results were obtained from different culture samples, and both of them exhibit similar patterns. Two other enzyme plate assays were obtained from different colonies, and both of them exhibit similar patterns.

Figure 6.

Secretion of negatively-charged proteins in pFD10 and pBD10. Western blottings were performed in a manner identical to that in the previous figures. A, Western blot of GFP. Both pFD10 and pBD10 exhibit an increase in protein secretion in the supernatant. B, Western blot of mannanase. Both pFD10 and pBD10 exhibit slight increases in mannase secretion. C, Western blot of MBP. The increased secretion ratio was observed in both pFD10 and pBD10. D, Western blot of thioredoxin. The signals were weak overall, but there was an increase in the secretion for both pFD10 and pBD10. Overall, the bands of more negatively-charged proteins in pBD10 appeared in slightly upward-shifted positions. Three other Western blot results for pDART and pBD10 were from different culture samples were obtained, whereas there were two other Western blot results for pDART and pFD10. All of them exhibit similar patterns.

Figure 7.

Secretion of TliA and GFP in pDART, pBD10, and pBR10. Two negatively-charged proteins, TliA and GFP, were inserted in the plasmids that attach nothing except the signal sequence (pDART), oligo-aspartate (pBD10), and oligo-arginine (pBR10). A, Western blot of TliA in these plasmids. TliA in pDART and pBD10 shows good secretion. However, the secretion was blocked when R10 was attached. B, enzyme plate assay of TliA in these plasmids. Secretion of TliA was blocked when it was inserted to pBR10. C, Western blot of GFP in these plasmids. Similarly, pDART and pBD10 showed good secretion, although secretion was blocked when R10 was attached.

Figure 8.

Secretion of GFP and supercharged GFPs. Western blotting was carried out in a manner identical to that of the previous figures. GFP(−30) exhibited a much higher secretion ratio than the original GFP, whereas GFP(+36) was not secreted at all despite its high expression. Note that the bands of the supercharged GFPs are also slightly shifted upwards. Two other Western blot results from different culture samples were obtained, and both of them exhibit similar patterns.

Figure 9.

Charge distribution in the structure of TliD, the ABC protein of the TliDEF complex. A, electron repulsion surface of the TliD monomer. Colored according to its surface electric potential, from blue (+7 k_BT/e) to red (−7 k_BT/e). The inner surface of the central channel is circled yellow. Note that the circled inner surface is highly positively charged. B, TliD homodimer, with one of the monomers presented in the ribbon model. Substrate entry window is circled green. C, TliD, residues with conserved positive or negative charges are colored blue and red, respectively. The conserved positive charge cluster at the midpoint of the channel's inner surface are circled yellow. The two α-helices that form substrate entry window are colored green. Among the two conserved positively-charged residues, Arg-316 (black arrow) sticks out to the pore. D, TliD dimer, seen from the periplasmic face. Positive charges are located in the middle of the channel (circled yellow), whereas negative charges are outside of the channel. E, schematic model of the TliD dimer, transporting a highly negatively charged recombinant polypeptide with the attached LARD3. The NBD and TMD of TliD are labeled accordingly. Note that the electric potential across the inner membrane (IM) is −150 mV, where the cytoplasm (CP) is more negative than the periplasm (PP). This potential difference also makes it more favorable to outward-transport negatively-charged proteins than positively-charged proteins. F, sequence mapping of the structures in C and D. Residues with high Bayesian conservation score (≥7) are highlighted in gray. Among them, the charged residues are colored as blue (+ charged) and red (− charged). Green and yellow boxes represent the substrate entry window and the channel's inner surface, respectively.