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A New Approach to Produce HIV-1 Envelope Trimers: BOTH CLEAVAGE AND PROPER GLYCOSYLATION ARE ESSENTIAL TO GENERATE AUTHENTIC TRIMERS.

AlSalmi W, Mahalingam M, Ananthaswamy N, Hamlin C, Flores D, Gao G, Rao VB - J. Biol. Chem. (2015)

Bottom Line: However, this antibody-based approach may not be as effective for the diverse HIV-1 strains with different epitope signatures.Uncleaved trimers entered aberrant pathways, resulting in hyperglycosylation, nonspecific cross-linking, and conformational heterogeneity.These studies established a broadly applicable HIV-1 trimer production system as well as generating new insights into their assembly and maturation that collectively bear on the HIV-1 vaccine design.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Biology, The Catholic University of America, Washington, D. C. 20064.

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Related in: MedlinePlus

Strep-tag II with an extended linker allows rapid purification of HIV-1 gp140 envelope trimers.A, schematic of gp140 expression cassette. PCMV, CMV promoter; CD5, secretion peptide; C1–C5, conserved domains; V1–V5, variable domains; black trees, glycans in gp120; blue trees, glycans in gp41; BGHpA, 3′ poly-A sequence of bovine growth hormone gene. The positions of the furin cleavage site, disulfide bond mutations (SOS), and I559P mutation are shown. B, tags attached to gp140 C terminus. The aa sequence of each tag is shown in the boxes. HRV-3C refers to human rhinovirus protease cleavage site. C, and D, reducing SDS-polyacrylamide gels showing protein patterns of the samples as indicated at the top. E, SDS gel of samples under reducing (+DTT) or non-reducing (−DTT) conditions. Red arrows, oligomers of gp120 or gp140 formed by nonspecific disulfide cross-linking. Blue arrows correspond to the ladder of gp41 ectodomain bands glycosylated to varying extents. F, Western blot using Strep-tag II-specific mAb. G, BN gel of Strep-Tactin-purified gp140 samples. Lanes labeled M, Mr markers. The molecular masses in kDa of the marker proteins are shown on the left. Gels in C, D, E, and G were stained with Coomassie Blue.
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Figure 2: Strep-tag II with an extended linker allows rapid purification of HIV-1 gp140 envelope trimers.A, schematic of gp140 expression cassette. PCMV, CMV promoter; CD5, secretion peptide; C1–C5, conserved domains; V1–V5, variable domains; black trees, glycans in gp120; blue trees, glycans in gp41; BGHpA, 3′ poly-A sequence of bovine growth hormone gene. The positions of the furin cleavage site, disulfide bond mutations (SOS), and I559P mutation are shown. B, tags attached to gp140 C terminus. The aa sequence of each tag is shown in the boxes. HRV-3C refers to human rhinovirus protease cleavage site. C, and D, reducing SDS-polyacrylamide gels showing protein patterns of the samples as indicated at the top. E, SDS gel of samples under reducing (+DTT) or non-reducing (−DTT) conditions. Red arrows, oligomers of gp120 or gp140 formed by nonspecific disulfide cross-linking. Blue arrows correspond to the ladder of gp41 ectodomain bands glycosylated to varying extents. F, Western blot using Strep-tag II-specific mAb. G, BN gel of Strep-Tactin-purified gp140 samples. Lanes labeled M, Mr markers. The molecular masses in kDa of the marker proteins are shown on the left. Gels in C, D, E, and G were stained with Coomassie Blue.

Mentions: Codon-optimized gp140 DNAs from JRFL-FD, SF162-FD, and CONPEP-FD were provided by Dr. Peter Kwong (Vaccine Research Center, National Institutes of Health). These DNAs contained the sequence corresponding to gp120 and gp41 ectodomain up to aa 683. In addition, they have the human CD5 secretion signal at the 5′-end, furin cleavage-resistant mutation SEKS at the junction of gp120 and gp41, and FD followed by the hexahistidine tag at the C terminus (49). Using the JRFL-FD as the starting template, a series of additional mutations were introduced. These include, for instance, SOSIP mutations (50, 51), stabilizing mutations (52), enhanced furin cleavage site RRRRRR (53), and various truncations shown in Fig. 2 and described under “Results.” The JRFL gp120 clone was also constructed from the same template by PCR amplification of the appropriate sequence corresponding to gp120.


A New Approach to Produce HIV-1 Envelope Trimers: BOTH CLEAVAGE AND PROPER GLYCOSYLATION ARE ESSENTIAL TO GENERATE AUTHENTIC TRIMERS.

AlSalmi W, Mahalingam M, Ananthaswamy N, Hamlin C, Flores D, Gao G, Rao VB - J. Biol. Chem. (2015)

Strep-tag II with an extended linker allows rapid purification of HIV-1 gp140 envelope trimers.A, schematic of gp140 expression cassette. PCMV, CMV promoter; CD5, secretion peptide; C1–C5, conserved domains; V1–V5, variable domains; black trees, glycans in gp120; blue trees, glycans in gp41; BGHpA, 3′ poly-A sequence of bovine growth hormone gene. The positions of the furin cleavage site, disulfide bond mutations (SOS), and I559P mutation are shown. B, tags attached to gp140 C terminus. The aa sequence of each tag is shown in the boxes. HRV-3C refers to human rhinovirus protease cleavage site. C, and D, reducing SDS-polyacrylamide gels showing protein patterns of the samples as indicated at the top. E, SDS gel of samples under reducing (+DTT) or non-reducing (−DTT) conditions. Red arrows, oligomers of gp120 or gp140 formed by nonspecific disulfide cross-linking. Blue arrows correspond to the ladder of gp41 ectodomain bands glycosylated to varying extents. F, Western blot using Strep-tag II-specific mAb. G, BN gel of Strep-Tactin-purified gp140 samples. Lanes labeled M, Mr markers. The molecular masses in kDa of the marker proteins are shown on the left. Gels in C, D, E, and G were stained with Coomassie Blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Strep-tag II with an extended linker allows rapid purification of HIV-1 gp140 envelope trimers.A, schematic of gp140 expression cassette. PCMV, CMV promoter; CD5, secretion peptide; C1–C5, conserved domains; V1–V5, variable domains; black trees, glycans in gp120; blue trees, glycans in gp41; BGHpA, 3′ poly-A sequence of bovine growth hormone gene. The positions of the furin cleavage site, disulfide bond mutations (SOS), and I559P mutation are shown. B, tags attached to gp140 C terminus. The aa sequence of each tag is shown in the boxes. HRV-3C refers to human rhinovirus protease cleavage site. C, and D, reducing SDS-polyacrylamide gels showing protein patterns of the samples as indicated at the top. E, SDS gel of samples under reducing (+DTT) or non-reducing (−DTT) conditions. Red arrows, oligomers of gp120 or gp140 formed by nonspecific disulfide cross-linking. Blue arrows correspond to the ladder of gp41 ectodomain bands glycosylated to varying extents. F, Western blot using Strep-tag II-specific mAb. G, BN gel of Strep-Tactin-purified gp140 samples. Lanes labeled M, Mr markers. The molecular masses in kDa of the marker proteins are shown on the left. Gels in C, D, E, and G were stained with Coomassie Blue.
Mentions: Codon-optimized gp140 DNAs from JRFL-FD, SF162-FD, and CONPEP-FD were provided by Dr. Peter Kwong (Vaccine Research Center, National Institutes of Health). These DNAs contained the sequence corresponding to gp120 and gp41 ectodomain up to aa 683. In addition, they have the human CD5 secretion signal at the 5′-end, furin cleavage-resistant mutation SEKS at the junction of gp120 and gp41, and FD followed by the hexahistidine tag at the C terminus (49). Using the JRFL-FD as the starting template, a series of additional mutations were introduced. These include, for instance, SOSIP mutations (50, 51), stabilizing mutations (52), enhanced furin cleavage site RRRRRR (53), and various truncations shown in Fig. 2 and described under “Results.” The JRFL gp120 clone was also constructed from the same template by PCR amplification of the appropriate sequence corresponding to gp120.

Bottom Line: However, this antibody-based approach may not be as effective for the diverse HIV-1 strains with different epitope signatures.Uncleaved trimers entered aberrant pathways, resulting in hyperglycosylation, nonspecific cross-linking, and conformational heterogeneity.These studies established a broadly applicable HIV-1 trimer production system as well as generating new insights into their assembly and maturation that collectively bear on the HIV-1 vaccine design.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Biology, The Catholic University of America, Washington, D. C. 20064.

Show MeSH
Related in: MedlinePlus