2. Successful Over-Expression of Transport Systems
2.1. SLC1
The SLC1 family includes seven members involved in amino acid traffic in cells. The family members are divided in two groups according to substrate specificity and transport mode
[12][151]. The first group (members A1, A2, A3, A6, and A7), known as EAATs (excitatory amino acid transporters), includes transporters with high affinity for the negatively charged amino acids glutamate and aspartate; the second group includes SLC1A4 and A5, known as ASCTs (alanine, serine, and cysteine transporters), involved in the traffic of several neutral amino acids in a broad set of tissues
[13][152]. The members of the SLC1 family possess eight membrane-spanning domains, are glycosylated, and contain between 524 and 574 amino acids. Even though the 3D-structures of the last two members have recently been solved by Cryo-EM using
P. pastoris as the expression host
[14][15][15,153], the ASCT2 protein was also produced in
E. coli [16][53]. The expression strategy employed the use of the Rosetta-gami2 strain, which joins the human tRNA supply and is involved in disulfide formation. For successful expression, a low temperature strategy had to be used to reduce protein toxicity. To achieve this, the pCOLD I vector—which carries the cold shock protein (csp), a promoter activated at low temperature—was used for cDNA cloning. Before the addition of 0.4 mM IPTG, a cold shock (10 min on ice and 40 min at 15 °C) was performed to improve the transcription of the ASCT2 mRNA and the stability of the 5′-UTR according to the feature of the cold-shock protein A promoter
[17][154]. To antagonize the protein toxicity observed as cell culture OD reduction, glucose was added to prevent leakiness, and the growth temperature post-induction was kept at 15 °C to reduce the basal metabolism.
2.2. SLC2
The SLC2 family includes 14 GLUT members involved in the transport of monosaccharides, polyols, and other small carbon compounds across eukaryotic cell membranes
[18][155]. The GLUT proteins contain about 500 amino acid residues, are glycosylated, and have 12 membrane-spanning domains. The human GLUT1 transporter, coded by the SLC2A1 gene, is the first example of a human transporter expressed in
E. coli [19][74]. The cDNA coding for hGLUT1 was cloned into a pGTSD12 plasmid with an upstream prokaryote-type ribosome binding site in a T7 promoter/T7 polymerase expression system. The strategy consisted of exploiting the insertion of the recombinant GLUT1 protein in the cell membrane of the SR425
E. coli strain, which was rid of all the bacterial glucose transporter genes (
ptsG,
ptsM,
gal) and transformed with the gene coding for the T7 RNA polymerase under the control of the heat-inducible pL promoter. In this case, the transport function of hGLUT1 could be assayed directly in the bacterial host.
14C-glucose transport inhibited by 2-deoxy-D-glucose and D-glucose, but not by L-glucose, was observed, confirming the expression of the target protein. In this system, the kinetics of transport were also studied, highlighting that—as observed in erythrocytes—glucose transport is inhibited by cytochalasin B and by mercuric chloride
[19][74].
2.3. SLC3
The SLC3 family includes two members—SLC3A1 (also named rBAT) and SLC3A2 (also named 4F2hc or CD98hc)—that share about 20% of their amino acid sequence identity
[20][156]. Both proteins are N-glycosylated: ~94 and ~85 kDa for the mature glycosylated forms of rBAT (685 aa) and 4F2hc (630 aa), respectively. The two proteins are type-II membrane N-glycoproteins with a single TMD and an intracellular N-terminus. They are characterized by a bulky extracellular C-terminus domain (50–60 kDa) that has been expressed in
E. coli. Being highly water soluble, this domain has been crystallized and its structure solved by X-ray diffraction
[21][157]. Even though these proteins are classified as members of the SLC superfamily, they are not directly involved in solute transport, but form heterodimers with some members of the SLC7 family, which are the subunits competent for transport
[22][158]. cDNA coding for the entirety of SLC3A2 was cloned in a pGEX-4T1 vector that includes an N-terminal GST tag. The corresponding protein could then be over-expressed in Rosetta(DE3)pLysS, probably due to the higher solubility of the chimeric protein (SLC3A2-GST) with respect to the sole SLC3A2. The chimeric protein was purified on a glutathione Sepharose 4B affinity column, then cleaved using thrombin treatment
[23][61].
2.4. SLC5
The SLC5 family includes 12 members that are sodium-dependent transporters involved in intestinal absorption (SLC5A1) or in the renal re-absorption (SLC5A2) of sugars
[24][159]. The sole member of this family expressed in
E. coli is SLC5A1
[25][126]. The use of a BL21 strain defective in the outer membrane protease (OmpT), together with low incubation temperatures (16 °C) and transcriptional regulation from the lac promoter/operator, have been crucial to reducing proteolytic degradation. Because bacterial cotransporters possess significantly shorter N-terminal hydrophilic extensions with respect to their eukaryote counterparts
[26][160], amino acid residues 12–28 were removed, promoting insertion in the
E. coli membrane. To recover the over-expressed protein and assay the transport function, SLC5A1 was solubilized with FosCholine-12 detergent, purified, and reconstituted in proteoliposomes in an active form
[25][126].
2.5. SLC6
This family includes 20 secondary active co-transporters with 12 membrane-spanning domains that utilize a chemiosmotic Na
+ gradient and/or Cl
− to couple the transport of their substrates across a membrane
[27][161]. The serotonin transporter (SLC6A4) was expressed and targeted to
E. coli membranes by combining codon optimization and tRNA supply in different strains and media
[28][57]. Another member of the SLC6 family, the SLC6A19 (also named B0AT1), was expressed in
E. coli exploiting human tRNA supplementation (BL21 CodonPlus RIL strain), combining a cold shock strategy (csp promoter, see SLC1A5) with a very low inducer concentration (10 μM) in the presence of 0.5% glucose
[16][53]. This protein is in complex with ACE2, constituting part of the receptor for the SARS-CoV-2 RBD proteins
[29][162]. The production of B0AT1 in
E. coli at a high yield can be useful for studying the interaction with compounds, which may have the potential for application as COVID-19 drugs
[30][31][163,164].
2.6. SLC7
The SLC7 family includes 13 members divided in two subfamilies: the cationic amino acid transporters (CATs, SLC7A1–4, and SLC7A14), and the light or catalytic subunits (L-type amino acid transporters LATs, and SLC7A5-13) of the heteromeric amino acid transporters (HATs); these are mostly exchangers with a broad spectrum of substrates, ranging from neutral to negatively charged amino acids
[20][156]. The members of this family differ in length—ranging from 470 amino acids for the SLC7A13 to 771 amino acids for the SLC7A14 members—and, consequently, in TM domains (12–14). SLC7A5 was over-expressed in the Rosetta(DE3)pLysS strain, (human tRNA supply) under standard conditions such as 4 h of 0.4 mM IPTG induction at 28 °C
[23][61]. The addition of an N-terminal 6His tag was crucial for IMAC purification of a protein that was refolded on a column and reconstituted into proteoliposomes. Several structure/function relationships were also defined thanks to the production of several mutants, with relevance to physiology and pathology
[32][33][34][165,166,167].
2.7. SLC17
The SLC17 family is a group of nine structurally related membrane proteins that mediate the transport of organic anions. SLC17A1–4s, also known as type-I phosphate transporters, are involved in the sodium-dependent transport of inorganic phosphate and other organic anions, such as urate and para-aminohippurate
[35][168]. The member SLC17A5, also known as sialin, catalyzes the lysosomal transport of sialic acid and acidic sugar, including glucuronic acid. The SLC17A6-8s (vGLUTs) localize to synaptic vesicles, but each appears to have a different distribution among other cell membranes where they are involved in glutamate transport
[36][169]. The SLC17A9 member (VNUT) is expressed in several tissues in mammals and is involved in vesicular nucleotide transport
[37][170]. The SLC17 family members share a similar topology, since they are predicted to have 12 TM domains with intracellular N- and C-
termini, as confirmed by the recently solved structure of the rat orthologous SLC17A6
[38][171]. Two out of nine members of the SLC17 family were expressed in
E. coli, exploiting a strategy that combines low temperature expression and membrane targeting using N-terminal and/or C-terminal fusion peptides, constituting 120 amino acids of the YbeL bacterial protein (named β- domain)
[39][66]. In particular, β- domains were added at both the N- and C-termini of the SLC17A5 and only at the C terminus of the SLC17A9 cDNAs. The fusion constructs were cloned in a pET-28a(+) vector. C43 cells were induced with 1 mM IPTG at 18 °C for 16 h in order to promote the insertion of the proteins in the membrane of the bacterial host. The proteins were purified using Ni-NTA chromatography and reconstituted in liposomes in a functionally active state
[39][66].
2.8. SLC18
The four members of the SLC18 family mediate the transport of neurotransmitters (SLC18A1-A3) or polyamines (SLC18B1)
[40][41][172,173]. Most computer-based predictions suggest 12 TMs with the N- and C-termini facing the intracellular milieu and a larger luminal loop between the first and second transmembrane domains. For the expression of the SLC18A3/VAChT, which mediates the transport of acetylcholine, the same strategy adopted for the members of the SLC17 family was used. The insertion in the C43(DE3) membrane was triggered by adding a YbeL tag at both the N- and C-termini of the protein after cloning in a pET-28a(+) vector. The induction of protein synthesis occurred at 18 °C for 16 h in the presence of 1 mM IPTG
[42][20]. The use of a 6His tag allowed purification via Ni-NTA affinity chromatography, followed by MALDI mass spectrometry analysis. Which confirmed the production of the target protein
[42][20].
2.9. SLC22
The SLC22 family is consists of at least 31 transporters expressed on both the apical and basolateral surfaces of epithelial cells, where they direct small-molecule transport between the body fluids and vital organs, such as the kidney, liver, heart, and brain
[43][44][174,175]. The family includes organic cation transporters (OCTs), novel organic cation transporters (OCTNs), and organic anion transporters (OATs) with different modes of transport. They have been defined as “drug transporters” due to their role in the absorption and excretion of drugs. These proteins share 12 α-helical TM domains, a large extracellular domain between TM1 and TM2, and a large intracellular domain between TM6 and TM7
[45][176]. Two members of the OCTN subfamily (SLC22A4 and SLC22A5) were over-expressed in
E. coli with different strategies
[46][47][46,62]. The cDNA coding for SLC22A4/OCTN1 was cloned in the pH6EX3 expression vector, and the Rosetta(DE3)pLysS strain was used for human tRNA supply. Protein synthesis was induced with 0.4 mM IPTG at 28 °C for 6 h and a 6His-tagged protein was purified and functionally reconstituted in proteoliposomes
[47][48][49][62,177,178]. After codon optimization, the use of the Lemo21 strain strongly increased the production of the target protein
[50][127]. The over-expression of OCTN1 and the production of several mutants allowed researchers to reveal the structure/function relationships between this transporter and the molecular basis of human diseases
[48][49][51][177,178,179]. Moreover, a specific antibody (anti-OCTN1) was produced using the over-expressed protein
[52][180]. Despite the high sequence similarity (86.5%), the same approach was not effective for SLC22A5/OCTN2. For the production of the protein, the use of an N-terminal GST tag was exploited, but the amount of protein recovered after expression in Rosetta(DE3)pLysS and tag removal was quite low
[46]. Interestingly, the conservative substitution of the second codon (R2K), introducing the statistically most present codon at the second position of
E. coli genes
[53][89], allowed the production of the target protein in a much larger amount with respect to the wild-type protein
[46].
2.10. SLC25
With its 53 members, the SLC25 family is the largest among the solute carrier families. Based on sequence similarities, human MCs cluster into many different clades, suggesting a large variety of transported substrates, among which are nucleotides, carboxylates, amino acids
[54][181]. Most MCs contain about 300 amino acids, with six TMs and the N- and C-termini protruding towards the cytosolic side of the inner membrane. For the expression of several members of the SLC25 family, neither human tRNA supply nor codon optimization was necessary (
Table 1). The most-used strategy for the expression of several members of the SLC25 family was based on inclusion body formation, obtained by combining a BL21(DE3) derivative strain with a pET or T7 derivative plasmid (
Table 1). In particular, the first protein over-expressed in a bacterial host, and then purified and assayed in an in vitro system, was the bovine oxoglutarate carrier, which was cloned in a pRUN vector
[55][114]. Then, the oxodicarboxylate (ODC, SLC25A21), and glutamate carrier 1 and 2 (SLC25A22 and SLC25A18, respectively) were also cloned in the pRUN plasmid. The corresponding proteins were expressed as inclusion bodies after culturing C0214 cells for 4.5 h in the presence of 0.4 mM IPTG at 37 °C, and reconstituted in proteoliposomes in an active form
[56][57][68,69]. The same method with a similar plasmid or strain was used for studying the carnitine/acylcarnitine translocase (CACT)
[58][67]; the ornithine/citrulline carrier (ORNT1)
[59][128]; the basic amino acid transporter (SLC25A29/ORNT3)
[60][129]; the S-adenosylmethionine transporter (SLC25A26)
[61][54]; the ATP-Mg/Pi transporters APC1 (SLC25A24) and APC3 (SLC25A25)
[62][72]; and the peroxisomal transporter of coenzyme A, FAD and NAD
+ (SLC25A17/PMP34)
[63]. This strategy was also used for the production of all the UCP isoforms (see
Table 1). Indeed, Ivanova et al. cloned all five UCPs in pET-21a(+) and expressed the corresponding proteins in BL21(DE3) as a 6His-tagged protein in inclusion bodies, after 3h of 1 mM IPTG induction at 37 °C
[64][56]. Following purification, the UCPs were reconstituted in stable, small, unilamellar vesicles, as confirmed by circular dichroism analysis
[64][56]. The same expression strategy was adopted for the expression of UCP2 and UCP3, which were refolded from inclusion bodies using a dialysis method
[65][66][65,123]. A completely different approach was used for the expression of the UCP1 protein, in which the insertion of the protein into the
E. coli membrane was obtained by adding the small periplasmic leader sequence (PelB), provided by the pET-26b(+) vector and culturing BL21 CodonPlus(DE3)-RIPL with an auto-induction method
[67][55]. The protein was extracted from the
E. coli membrane, purified, and reconstituted into phospholipid bilayers in an active form
[68][182]. In addition to
E. coli,
L. lactis was also used for the expression of the regulatory domains of aralar1 (SLC25A12) and citrin (SLC25A13). In particular, the N- and C-terminal domain of both proteins were cloned in the pNZ8048 expression vector, produced as 8His-tagged proteins in
L. lactis NZ9000, purified using a nickel-Sepharose high-performance column, and crystallized
[69][13].
Table 1.
Strategy adopted for membrane protein expression.
Protein/Alias |
Plasmid |
Tag |
Strain |
Strategy |
Function |
Reference |
SLC1A5/ASCT2 |
pCOLD-I |
N-Ter 6His |
Rosetta-gami2 |
Low temperature/glucose |
ND |
[16][53] |
SLC2A1/GLUT1 |
pGTSD12 |
None |
SR425 |
PL promoter, membrane insertion |
M |
[19][74] |
SLC3A2/4F2hc |
pGEX-4T1 |
N-Ter-GST |
Rosetta(DE3)pLysS |
28 °C |
ND |
[23][61] |
SLC5A1/SGLT1 |
pTMH-6FH |
FLAG |
BL-21 |
0.3 mM IPTG, 16 °C, 5 h |
P |
[25][126] |
SLC6A4/SERT |
TAGZyme pQE-2 |
N-Ter 8His |
BL21 CodonPlus (DE3) RP |
Recovery from membrane |
S |
[70][58] |
SLC6A19/B0AT1 |
pCOLD-I |
N-Ter 6His |
BL21 codonPlus RIL |
Low temperature/low IPTG/glucose |
ND |
[16][53] |
SLC7A5/LAT1 |
pH6EX3 |
N-Ter 6His |
Rosetta(DE3)pLysS |
0.4 mM IPTG, 28 °C, 4 h |
P |
[23][61] |
SLC17A5/Sialin |
pET-28a(+) |
N-Ter and C-Ter β domain |
C43(DE3) |
Membrane insertion, 1 mM IPTG, 18 °C, 16 h |
M |
[39][66] |
SLC17A9/VNUT |
pET-28a(+) |
N-Ter β domain |
C43(DE3) |
Membrane insertion, 1 mM IPTG, 18 °C, 16 h |
M |
[39][66] |
SLC18A3/VAChT |
pET-28a(+) |
N- and C-ter YbeL |
C43(DE3) |
Membrane insertion, 1 mM IPTG, 18 °C, 16 h |
M |
[42][20] |
SLC22A4/OCTN1 |
pH6EX3 |
N-Ter 6His |
Rosetta(DE3)pLysS |
0.4 mM IPTG 28 °C, 6 h |
P |
[47][62] |
SLC22A4/OCTN1 |
pH6EX3 |
N-Ter 6His |
Lemo21(DE3) |
Codon optimization, 0.4 mM IPTG 28 °C, 6 h |
P |
[50][127] |
SLC22A5/OCTN2 |
pET-21a(+) |
C-Ter 6His |
Rosetta(DE3)pLysS |
R2K mutation |
N |
[46] |
SLC22A5/OCTN2 |
pET-41a(+) |
GST |
Rosetta(DE3)pLysS |
28 °C, 6 h |
N |
[46] |
SLC25A7/UCP1 |
pET-26b(+) |
PelB/6His/TEV |
BL21 CodonPlus (DE3)-RIPL |
Auto-induction method |
S,P |
[67][55] |
SLC25A7/UCP1 |
pET-21a(+) |
N-Ter 6His |
BL21(DE3) |
1 mM IPTG, 37 °C, 3 h |
S,P |
[64][56] |
SLC25A8/UCP2 |
pET-21a(+) |
N-Ter 6His |
BL21(DE3) |
1 mM IPTG, 37 °C, 3 h |
S,P |
[64][56] |
SLC25A8/UCP2 |
pET-21a(+) |
None |
BL21(DE3) |
1 mM IPTG, 30 °C, 6 h |
P |
[66][123] |
SLC25A8/UCP2 |
pMW172 |
None |
C41 |
2 h, 1 mM IPTG at 37 °C |
ND |
[65] |
SLC25A9/UCP3 |
pET-21d(+) |
N-Ter 6His |
BL21(DE3) |
1 mM IPTG, 37 °C, 3 h |
S,P |
[64][56] |
SLC25A9/UCP3 |
pET-21a(+) |
None |
BL21(DE3) |
1 mM IPTG, 30 °C, 6 h |
P |
[66][123] |
SLC25A9/UCP3 |
pET-24a(+) |
None |
BL21(DE3) |
1 mM IPTG, 37 °C, 2 h |
P |
[71][122] |
SLC25A12/Aralar1 |
pNZ8048 |
N-Ter 8His |
L. lactis NZ9000 |
Codon optimization |
C,P |
[69][13] |
SLC25A13/Citrin |
pNZ8048 |
N-Ter 8His |
L. lactis NZ9000 |
Codon optimization |
C,P |
[69][13] |
SLC25A14/UCP5 |
pET-21a(+) |
N-Ter 6His |
BL21(DE3) |
1 mM IPTG, 37 °C, 3 h |
S,P |
[64][56] |
SLC25A15/ORNT1 |
pET-21a(+) |
C-Ter 6His |
C0214 |
0.4 mM IPTG, 28 °C 4 h |
P |
[59][128] |
SLC25A17/ PMP34 |
pET-21b |
T7 |
Rosetta-gami B |
30 °C inclusion bodies |
P |
[63] |
SLC25A18/GC2 SLC25A22/GC1 |
pRUN |
None |
C0214(DE3) |
0.4 mM IPTG, 37 °C 4.5 h |
P |
[57][69] |
SLC25A20/CACT |
pMW7 |
None |
C0214(DE3) |
0.4 mM IPTG, 37 °C 4 h |
P |
[58][67] |
SLC25A21/ODC |
pRUN |
None |
C0214(DE3) |
0.4 mM IPTG, 37 °C 4.5 h |
P |
[56][68] |
SLC25A24/APC1 SLC25A25/APC3 |
pQE30 |
N-Ter 6His |
M15(pREP4) |
Manufacturer’s instructions |
P |
[62][72] |
SLC25A26/SAMC |
pRUN |
None |
BL-21 CodonPlus(DE3)-RIL |
0.4 mM IPTG, 37 °C 4.5 h |
P |
[61][54] |
SLC25A27/UCP4 |
pET-21a(+) |
N-Ter 6His |
BL21CodonPlus (DE3)-RIPL |
1 mM IPTG, 37 °C, 3 h |
S,P |
[64][56] |
SLC25A29/ORNT3 |
pRUN |
None |
Rosetta-gami B(DE3) |
37 °C |
P |
[60][129] |
SLC29A1/ENT1 |
pHAT20 |
3xFLAG |
BL21(DE3) |
<25 °C |
|
[72][107] |
SLC30A8/ZnT8 |
pTrxFus |
N-Ter Thioredoxin |
GI724 |
Inclusion bodies |
ND |
[73][45] |
SLC30A8/ZnT8 |
pTrxFus |
N-Ter Thioredoxin |
GI698 |
Intracellular soluble fraction |
ND |
[73][45] |
SLC35C1/FUCT1 |
pJOE2702 |
ompA/FLAG |
BW25113(DE3) |
Codon optimization, membrane insertion |
M |
[74][71] |
SLC35F3 |
pZE12luc |
None |
MW25113 |
Membrane insertion |
M |
[75][51] |
SLC38A9 |
pH6EX3 |
N-Ter 6His |
Lemo21(DE3) |
Codon optimization, 39 °C, 2 h |
P |
[76][70] |
SLC52A2/RFVT2 |
pH6EX3 |
N-Ter 6His |
Rosetta(DE3) |
Codon optimization |
P |
[77][78][59,60] |
ABCB10 |
pET19b |
N-Ter 6His |
Rosetta2, BL21-CodonPlus (DE3)-RIPL |
Codon optimization |
L,S |
[79][52] |
MDR1 |
pPOW-B2 |
None |
UT5600 |
Membrane insertion |
M |
[80][73] |
hTRPV3 |
pWaldo–GFPe |
GFP |
BL21(DE3)-R3- pRARE 2 |
1 mM IPTG, TB medium, at 18 °C, |
B |
[81][64] |
EAG1 |
pRSET-A |
N-Ter 6His |
BL21(DE3) |
1 mM IPTG, 37 °C, 4 h |
ND |
[82][130] |
MiRP1 |
pET-21b |
C-Ter 6His |
BL21(DE3) CodonPlus RP |
1 mM IPTG, 37 °C, 4 h |
S |
[28][57] |
ROMK1 |
pET-28a(+) |
Several tags |
BL21(DE3)-pLysS |
Codon optimization, membrane insertion |
L |
[83][124] |
CLIC4 |
pET-22b(+) |
C-Ter 6His |
BL21(DE3) |
1 mM IPTG, 37 °C, 6 h |
C |
[84][131] |
MICU1 |
pGEX-6p-1 |
N-Ter GST |
BL21 DE3 |
0.5 mM IPTG, 16 °C, 20 h |
S |
[85][50] |
MICU2-NΔ84-CΔ28 |
pET-28a(+) |
N-Ter 6His |
BL21 DE3 |
0.5 mM IPTG, 16 °C, 20 |
S |
[85][50] |
hVDAC1 |
pET-21a(+) |
C-Ter 6His |
BL21(DE3) |
1 mM IPTG, 37 °C, 3 h |
N,PP |
[86][87][121,132] |
hVDAC1 hVDAC2 |
PDS56/RBII |
C-Ter 6His |
M15(pREP4) |
1 mM IPTG, 37 °C, 5–6 h |
PP,S |
[88][133] |
hVDAC3 |
pET-21a(+) |
C-Ter 6His |
BL21(DE3) |
1 mM IPTG, 37 °C, 3 h |
PP |
[89][44] |