Table 1.
Rarely used codons by E. coli in genes of HIV-1.
Table 2.
Expression vectors engineered in this study.
Table 3.
Oligonucleotides used to construct and verify pSA-HNef-6His, pSA-HNef-6His-RIL, pSA-Hp24-6His-RIL, pSA-HVif-6His-RIL, and pSA-C6His-RIL expression vectors.
Fig 1.
Schematic representation of expression vector pSA-HNef-6His.
The nef gene was PCR amplified from pNL4.3 plasmid, restricted with NdeI/ SacI, and ligated into pSA-6His vector at the same sites.
Fig 2.
SDS-PAGE and immunoblot analysis of recombinant Nef.
NiCo21(DE3) E. coli were transformed with pSA-HNef-6His vector and grown overnight from a single colony at 30°C in LB broth supplemented with 100 μg/ml ampicillin. The cultures were diluted 100-fold in the same medium and grown to mid-log phase (OD600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.4 mM. The induced cells were grown for another 6 h at 30°C and stored on ice. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by (A) Coomassie staining and (B) immuno-blotting. Lanes: M, PageRuler Prestained Protein Ladder Plus; WCL, whole cell lysate; IS, insoluble fraction; S, soluble fraction; M1, MagicMark XP Western Protein Standard. Recombinant Nef was produced in the induced cells but not in the un-induced controls, and mainly present in the soluble fraction.
Fig 3.
Optimization of IPTG concentration and induction temperature.
(A) For optimization of IPTG concentration, the overnight cultures of pSA-HNef-6His-transformed NiCo21(DE3) were diluted 1:100 in LB+Amp (100 μg/ml) and grown to mid-log phase (OD600 ~0.5–0.6). The cultures were then induced with varying concentrations (0, 0.05, 0.1, 0.2, 0.3, and 0.4 mM) of IPTG and grown for another 6 h at 30°C. Nine microliters of samples were mixed with 4x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by immuno-blotting. A concentration of 0.05 mM IPTG (red arrow) was sufficient to induce high level Nef expression. (B) For optimal induction temperature, the overnight cultures of pSA-HNef-6His-transformed NiCo21(DE3) were diluted 1:100 in LB+Amp (100 μg/ml) and grown to mid-log phase (OD600 ~0.5–0.6). The cultures were then induced with 0.05 mM of IPTG and grown at 30, 22, and 18°C for 6, 12, and 16 h respectively. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining. Nef expression was similar at 30 and 22°C but lower at 18°C. Lanes: M, BenchMark Pre-stained protein ladder; M1, PageRuler Prestained Protein Ladder Plus; 1, un-induced NiCo21 at 30°C (soluble); 2, IPTG-induced NiCo21 at 30°C (insoluble); 3, IPTG-induced NICo21 at 30°C (soluble); 4, un-induced NiCo21 at 22°C (soluble); 5, IPTG-induced NiCo21 at 22°C (insoluble); 6, IPTG-induced NiCo21 at 22°C (soluble); 7, un-induced NiCo21 at 18°C (soluble); 8, IPTG-induced NiCo21 at 18°C (insoluble); 9, IPTG-induced NiCo21 at 18°C (soluble).
Fig 4.
Effect of rare tRNA supplementation on Nef expression in NiCo21(DE3) E. coli.
(A) ORF coding for HIV-1 nef gene was subjected to rare codon analysis using an online tool ‘Rare Codon Calculator (RaCC)’ http://nihserver.mbi.ucla.edu/RACC/. Three rare codons encoding arginine and one rare codon encoding leucine are present at 17 amino acid positions in Nef ORF. (B) NiCo21(DE3) E. coli were individually transformed with rare tRNA-expressing helper plasmids (pACYC-RIL, pRARE2, pRARE2-lysS) and selected on LB+Cam. These bacteria were then made competent, transformed with pSA-HNef-6His, and selected on LB+Cam+Amp plates. Cultures were grown overnight from a single colony at 30°C in LB+Cam+Amp. The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (OD600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05 mM. The induced cells were grown for another 12 h at 22°C and stored on ice. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel analyzed by Coomassie staining. Expression of rare codon tRNA genes resulted in high level expression of Nef. Lanes: M, PageRuler Prestained Protein Ladder Plus; WCL, whole cell lysate; IS, insoluble fraction; S, soluble fraction.
Fig 5.
Schematic representation of expression vector pSA-HNef-6His-RIL.
A. Map of pSA-HNef-6His-RIL vector with rare tRNA genes array. B. Arrangement of rare tRNA genes in clockwise and counter clockwise orientation.
Fig 6.
Expression of Nef from pSA-HNef-6His vectors with or without rare tRNA genes array.
To evaluate the effect of the introduction of rare tRNA genes array in pSA-HNef-6His vector, the NiCo21 E. coli were individually transformed with pSA-HNef-6His, pSA-HNef-6His-RIL(CW), and pSA-HNef-6His-RIL(CCW) and Nef expression was compared with NiCo21 E. coli co-transformed with pSA-HNef-6His and rare tRNA expressing pACYC-RIL vectors. The cultures were grown overnight at 30°C in LB broth containing Amp (for bacteria harboring pSA-HNef-6His or pSA-HNef-6His-RIL) or Amp+Cam (for bacteria harboring pSA-HNef-6His + pACYC-RIL). The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (OD600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05 mM. The induced cells were grown for another 12 h at 22°C and stored on ice. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 15% SDS-PAGE gel and analyzed by (A) Coomassie staining, (B) immuno-blotting and (C) band densitometry. Nef expression in NiCo21 transformed with pSA-HNef-6His-RIL (CW/CCW) was comparable with NiCo21 co-transformed with pSA-HNef-6His and pACYC-RIL. A prominent band running at 25 kDa (red arrows) appears in NiCo21 harboring pSA-HNef-6His/ pACYC-RIL. This is most probably chloramphenicol acetyltransferase monomer. Higher baseline expression for un-induced cultures were noted in NiCo21 harboring pSA-HNef-6His-RIL(CCW) (see green arrows). This suggests that ileY tRNA gene most probably doesn’t contain a transcription terminator downstream and the baseline expression of Nef is a result of transcriptional read-through of mRNA synthesis from ileY promoter.
Fig 7.
Schematic representation of expression vectors pSA-HP24/Vif-6His-RIL.
Map shows the vector containing HIV-1 p24 or vif genes cloned downstream T7 promoter.
Fig 8.
Expression of HIV-1 P24 from pSA-HP24-6His vectors with or without rare tRNA genes array.
To evaluate the effect of the introduction of rare tRNA genes array in pSA-HP24-6His vector, the NiCo21(DE3) E. coli were individually transformed with pSA-HP24-6His, and pSA-HP24-6His-RIL and P24 expression was compared with NiCo21(DE3) E.coli co-transformed with pSA-Hp24-6His and rare tRNA expressing pACYC-RIL vectors. The cultures were grown overnight at 30°C in LB broth containing Amp (for bacteria harboring pSA-HP24-6His or pSA-HP24-6His-RIL) or Amp+Cam (for bacteria harboring pSA-HP24-6His + pACYC-RIL). The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C and stored on ice. Three microliters of samples were mixed with 4X loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining (A), immuno-blotting (B), and band densitometery (C). The P24 expression in NiCo21 transformed with pSA-HP24-6His-RIL was comparable with NiCo21 co-transformed with pSA-HP24-6His and pACYC-RIL.
Fig 9.
Expression of HIV-1 Vif from pSA-HVif-6His vectors with or without rare tRNA genes array.
To evaluate the effect of the introduction of rare tRNA genes array in pSA-HVif-6His vector, the NiCo21(DE3) E. coli were individually transformed with pSA-HVif-6His, and pSA-HVif-6His-RIL and Vif expression was compared with NiCo21(DE3) E.coli co-transformed with pSA-HVif-6His and rare tRNA expressing pACYC-RIL vectors. The cultures were grown overnight at 30°C in LB broth containing Amp (for bacteria harboring pSA-HVif-6His or pSA-HVif-6His-RIL) or Amp+Cam (for bacteria harboring pSA-HVif-6His + pACYC-RIL). The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C and stored on ice. Three microliters of samples were mixed with 4X loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining (A), immuno-blotting (B), and band densitometery (C). The Vif expression in NiCo21(DE3) transformed with pSA-HVif-6His-RIL was comparable with NiCo21 co-transformed with pSA-HVif-6His and pACYC-RIL.
Fig 10.
Production and purification of HIV-1 Nef and P24.
NiCo21(DE3) transformed with pSA-HNef/P24-6His, pSA-HNef/P24-6His/pACYC-RIL, and pSA-HNef/P24-6His-RIL were grown overnight from a single colony at 30°C in super broth (SB) containing appropriate antibiotic(s). The cultures were then diluted to 0.05 OD600 in the same medium and appropriate antibiotics(s). Cultures were grown (at 30°C) to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C. A small aliquot (100μL) of culture was removed every hour, diluted 10 fold in the same medium and cell density was measured at OD600 and plotted. A. Growth kinetics of cultures expressing HIV-1 Nef from various expression vectors/combinations. B. Comparison of Nef production/purification from cultures expressing Nef from pSA-HNef-6His/pACYC-RIL with those expressing Nef from pSA-HNef-6His-RIL vector. C. Growth kinetics of cultures expressing HIV-1 P24 from various expression vectors/combinations. D. Comparison of P24 production/purification from cultures expressing P24 from pSA-HP24-6His/pACYC-RIL with those expressing P24 from pSA-HP24-6His-RIL vector.
Fig 11.
Schematic representation of expression vectors pSA-C6His-RIL.
Map shows multiple cloning site with eight unique restriction enzyme sites for facile cloning of heterologous genes.