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Bira enzyme

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The BirA enzyme is a biotin-protein ligase that catalyzes the specific biotinylation of a target protein or peptide sequence. It is commonly used in various biotechnological applications that require the covalent attachment of biotin to recombinant proteins or other molecules.

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Lab products found in correlation

Galanthus nivalis lectin, by Vector Laboratories (3 mentions) Sephadex g 25 pd10 columns, by GE Healthcare (3 mentions) 293fectin, by Thermo Fisher Scientific (2 mentions) Superose 6 10 300 gl column, by GE Healthcare (2 mentions) Freestyle medium, by Thermo Fisher Scientific (2 mentions) Methyl α d mannopyranoside mmp, by Merck Group (2 mentions) Bicinchonic acid based assay, by Thermo Fisher Scientific (2 mentions) Hek293f, by Thermo Fisher Scientific (2 mentions) Freestyle 293 f cells, by Thermo Fisher Scientific (2 mentions) Streptavidin bound apc, by BioLegend (2 mentions) Extravidin pe, by Merck Group (2 mentions) Hiload 16 600 superdex pg200, by GE Healthcare (1 mentions) Vivaflow 50, by Sartorius (1 mentions) Quikchange multi kit, by Agilent Technologies (1 mentions) Zeba spin column, by Thermo Fisher Scientific (1 mentions) Amicon ultra 10 000 kda spin columns, by Merck Group (1 mentions) Adenosine triphosphate (atp), by Avidity (1 mentions) Biacore evaluation software, by GE Healthcare (1 mentions) Biacore s200, by GE Healthcare (1 mentions) Hek293f cell, by Thermo Fisher Scientific (1 mentions)

20 protocols using bira enzyme

1

Purification of Engineered HIV-1 Envelope Glycoproteins

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BG505 SOSIP.664 gp140, BG505 SOSIP.664-His gp140, and BG505 SOSIP.664-avi gp140 were expressed in HEK293F (Invitrogen) as described previously14 (link). Briefly, HEK293F cells were maintained in FreeStyle medium (Invitrogen). For gp140 trimer production, HEK293F cells were seeded at a density of 0.5×106/mL. After 24 h, cells were transfected with 1 mg of 293Fectin (Invitrogen) with 300 μg of Env plasmid and 75 μg of furin plasmid in OPTI-MEM according to the manufacturer’s protocol. Supernatants were purified using a Galanthus nivalis lectin (Vector Labs) column and protein was eluted with 1M methyl-α-D-mannopyranoside (MMP, Sigma). Following buffer exchange into PBS, only trimers with AviTags were in vitro biotinylated using the BirA enzyme according to the manufacturer protocol (Avidity). The affinity-purified Env proteins were further purified to size homogeneity using size exclusion chromatography (SEC) on a Superose 6 10/300 GL column (GE Healthcare) in PBS. The trimer fractions were collected and pooled and protein concentrations were determined using either a bicinchonic acid-based assay (Thermo Scientific) or UV280 absorbance using theoretical extinction coefficients.
Sok D., Le K.M., Vadnais M., Saye-Francisco K., Jardine J.G., Torres J., Berndsen Z.T., Kong L., Stanfield R., Ruiz J., Ramos A., Liang C.H., Chen P.L., Criscitiello M.F., Mwangi W., Wilson I.A., Ward A.B., Smider V.V, & Burton D.R. (2017). Rapid elicitation of broadly neutralizing antibodies to HIV by immunization in cows. Nature, 548(7665), 108-111.
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2

Purification of Engineered HIV-1 Envelope Glycoproteins

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BG505 SOSIP.664 gp140, BG505 SOSIP.664-His gp140, and BG505 SOSIP.664-avi gp140 were expressed in HEK293F (Invitrogen) as described previously14 (link). Briefly, HEK293F cells were maintained in FreeStyle medium (Invitrogen). For gp140 trimer production, HEK293F cells were seeded at a density of 0.5×106/mL. After 24 h, cells were transfected with 1 mg of 293Fectin (Invitrogen) with 300 μg of Env plasmid and 75 μg of furin plasmid in OPTI-MEM according to the manufacturer’s protocol. Supernatants were purified using a Galanthus nivalis lectin (Vector Labs) column and protein was eluted with 1M methyl-α-D-mannopyranoside (MMP, Sigma). Following buffer exchange into PBS, only trimers with AviTags were in vitro biotinylated using the BirA enzyme according to the manufacturer protocol (Avidity). The affinity-purified Env proteins were further purified to size homogeneity using size exclusion chromatography (SEC) on a Superose 6 10/300 GL column (GE Healthcare) in PBS. The trimer fractions were collected and pooled and protein concentrations were determined using either a bicinchonic acid-based assay (Thermo Scientific) or UV280 absorbance using theoretical extinction coefficients.
Sok D., Le K.M., Vadnais M., Saye-Francisco K., Jardine J.G., Torres J., Berndsen Z.T., Kong L., Stanfield R., Ruiz J., Ramos A., Liang C.H., Chen P.L., Criscitiello M.F., Mwangi W., Wilson I.A., Ward A.B., Smider V.V, & Burton D.R. (2017). Rapid elicitation of broadly neutralizing antibodies to HIV by immunization in cows. Nature, 548(7665), 108-111.
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3

Purification of HIV-1 Envelope Glycoproteins

Cited 3 times
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BG505 SOSIP.664, BG505 SOSIP.664-D7324 tag, BG505 SOSIP.664-AviTag, BG505 SOSIP.v4.1, and BG505 SOSIP.v5.2 were expressed in HEK293F cells and purified with either PGT145 or 2G12 affinity chromatograph followed by size exclusion chromatography (SEC) using a HiLoad 16/600 Superdex pg200 (GE Healthcare) as described previously [7 (link),8 (link),13 (link)]. Monomeric gp120 proteins (AviTag or D7324 tagged) were purified using a Galanthus nivalis lectin (Vector Labs) column. The Avi-tagged proteins were biotinylated using the BirA enzyme (Avidity) according to the manufacturer’s protocol. The resulting biotinylated proteins are referred to using the descriptor AviB.
Cottrell C.A., van Schooten J., Bowman C.A., Yuan M., Oyen D., Shin M., Morpurgo R., van der Woude P., van Breemen M., Torres J.L., Patel R., Gross J., Sewall L.M., Copps J., Ozorowski G., Nogal B., Sok D., Rakasz E.G., Labranche C., Vigdorovich V., Christley S., Carnathan D.G., Sather D.N., Montefiori D., Silvestri G., Burton D.R., Moore J.P., Wilson I.A., Sanders R.W., Ward A.B, & van Gils M.J. (2020). Mapping the immunogenic landscape of near-native HIV-1 envelope trimers in non-human primates. PLoS Pathogens, 16(8), e1008753.
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4

Production and Purification of HLA-E Complexes

Cited 1 time
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Both canonical and Tyr84Cys mutated HLA-E*01:03 heavy chains were expressed in E. coli BL21 (DE3) pLysS competent bacterial cells (Promega, UK) as inclusion bodies and purified according to methods described previously(1 (link), 15 (link), 21 ). Conventional and Tyr84Cys mutated HLA-E*01:03 heavy chains were refolded using standard MHC refolding methods (1 (link), 32 (link)). Following concentration using a Vivaflow 50 (Sartorius, Germany) and Ultra-15 10-kDa cut-off centrifugal units (Sartorius, UK), the samples were subsequently buffered exchanged, using Sephadex G-25 PD10 columns (GE Healthcare, UK) into 10mM Tris for overnight AviTAG biotinylation using the BirA enzyme (Avidity, USA) according to the manufacturer’s instructions. Correctly refolded complexes were purified by size exclusion fast protein liquid chromatography (FPLC) into 20mM Tris pH8 and 100mM NaCl buffer using a HiLoad 16/600 Superdex 75pg column. Correctly folded β2m-HLA-E*01:03-peptide complexes were retrieved, concentrated to 2mg/mL and snap frozen for subsequent tetramer generation.
Yang H., Rei M., Brackenridge S., Brenna E., Sun H., Abdulhaqq S., Liu M.K., Ma W., Kurupati P., Xu X., Cerundolo V., Jenkins E., Davis S.J., Sacha J.B., Früh K., Picker L.J., Borrow P., Gillespie G.M, & McMichael A.J. (2021). HLA-E restricted Gag specific CD8+ T cells can suppress HIV-1 infection, offering vaccine opportunities. Science immunology, 6(57), eabg1703.
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5

Biotinylation and Tetrameric Presentation of Peptide-HLA Complexes

Cited 2 times
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BirA substrate peptide (BSP)-tagged forms of the A*24:02 heavy chain and β2 microglobulin (β2m) were expressed, refolded with peptides (Nef134-10 [RYPLTFGWCF], Nef134-10(Y135F) [RFPLTFGWCF], Nef134-8 [RYPLTFGW], Nef134-8(Y135F) [RFPLTFGW]) (Sigma-Genosys) and purified as described [20 (link), 22 (link)]. BSP-tagged peptide-HLA complexes (pHLAs) were biotinylated by BirA enzyme (Avidity) and tetramerised by phycoerythrin (PE)- or allophycocyanin (APC)-labelled streptavidin.
Katoh J., Kawana-Tachikawa A., Shimizu A., Zhu D., Han C., Nakamura H., Koga M., Kikuchi T., Adachi E., Koibuchi T., Gao G.F., Brumme Z.L, & Iwamoto A. (2016). Rapid HIV-1 Disease Progression in Individuals Infected with a Virus Adapted to Its Host Population. PLoS ONE, 11(3), e0150397.
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6

Production and Purification of HIV-1 Env Trimers

Cited 2 times
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BG505 SOSIP.664-Avi gp140, BG505 SOSIP.664-His and other HIV Env trimers were expressed in FreeStyle 293F cells (ThermoFisher) as described previously (Walker et al., 2011 (link)). Supernatants were purified by affinity chromatography using a PGT145 column. Trimer with AviTags were biotinylated in vitro using the BirA enzyme (Avidity) according to the manufacturer’s instructions. The affinity-purified trimers were purified by size exclusion using a HiLoad 26/600 Superdex 200 pg column as described previously (Lee et al., 2017 (link)).
Zhao F., Joyce C., Burns A., Nogal B., Cottrell C.A., Ramos A., Biddle T., Pauthner M., Nedellec R., Qureshi H., Mason R., Landais E., Briney B., Ward A.B., Burton D.R, & Sok D. (2020). Mapping Neutralizing Antibody Epitope Specificities to an HIV Env Trimer in Immunized and in Infected Rhesus Macaques. Cell Reports, 32(10), 108122.
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7

Recombinant HIV-1 Glycoprotein Production

Cited 2 times
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The BG505 SOSIP.v5.2 and BG505 SOSIP.v5.2-Avitag plasmids were generously provided by Dr. Marit van Gils31 (link). Site-direct mutagenesis (QuikChange Multi kit, Agilent) was used create the plasmids encoding the Prime, Boost#1, Boost#2, Boost#3, Prime-Avitag, and Boost#2-Avitag. Specific mutations relative to BG505 SOSIP.v5.2 are listed in Table S5. Immunogens and Avi-tagged probes were produced in HEK293F cells and purified using PGT145 affinity chromatography followed size-exclusion chromatography as described previously59 (link). The Avi-tagged proteins were biotinylated using the BirA enzyme (Avidity) according to the manufacturer’s protocol. The resulting biotinylated proteins are referred to using the descriptor AviB.
Cottrell C.A., Pratap P.P., Cirelli K.M., Carnathan D.G., Enemuo C.A., Antanasijevic A., Ozorowski G., Sewall L.M., Gao H., Greene K.M., Allen J.D., Ngo J.T., Choe Y., Nogal B., Silva M., Bhiman J., Pauthner M., Irvine D.J., Montefiori D., Crispin M., Burton D.R., Silvestri G., Crotty S, & Ward A.B. (2023). Focusing antibody responses to the fusion peptide in rhesus macaques. bioRxiv.
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8

HLA-E Peptide Biotinylation and Tetramer Generation

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HLA-E-peptide samples requiring biotinylation were subsequently buffered exchanged on Sephadex G-25 PD10 columns (GE Healthcare, UK) into 10mM Tris buffer using commercially available BirA enzyme (Avidity, USA) following the manufacturer's instructions. Following overnight biotinylation, protein samples were subsequently purified into 20mM Tris pH8,100mM
NaCl buffer or PBS on a HiLoad 16/600 Superdex 75pg column using an AKTA size exclusion fast protein liquid chromatography (FPLC) system. Correctly folded β2m-HLA-E*01:03peptide complexes were subsequently concentrated to 2mg/mL and snap frozen.
HLA-E*01:03 tetramers were generated via conjugation to various fluorescent labels including Extravidin-PE (Sigma), Streptavidin-bound APC (Biolegend, San Diego) or BV421 (Biolegend, San Diego) at a Molar ratio of 4:1 as previously described (Braud et al., 1998) .
Li D., Brackenridge S., Walters L.C., Swanson O., Harlos K., Rozbeský D., Cain D.W., Wiehe K., Scearce R.M., Barr M., Mu Z., Parks R., Quastel M., Edwards R.J., Wang Y., Rountree W., Saunders K.O., Ferrari G., Borrow P., Jones E.Y., Alam S.M., Azoitei M.L., Gillespie G.M., McMichael A.J., & Haynes B.F. (2020). Mouse and Human Antibodies that Bind HLA-E-Leader Peptide Complexes and Enhance NK Cell Cytotoxicity.
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9

Protein Biotinylation for Functional Analysis

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Monomeric fractions of refolded proteins were biotinylated at the specific lysine residue in the Avitag sequence (GGGLNDIFEAQKIEWHE), using BirA enzyme (Avidity, LLC) overnight at 4°C. Excess biotin was removed and biotinylation buffer was exchanged to 1X PBS, pH = 7.4 using Zeba spin columns (Thermo Fisher Scientific Inc, IL, USA). Biotinylation was assessed by incubating the proteins with streptavidin and analyzing their change in mobility (“gel-shift”) on 4–20% polyacrylamide gels.
Sharma P., Wang N., Chervin A.S., Quinn C.L., Stone J.D, & Kranz D.M. (2015). A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins. PLoS ONE, 10(8), e0135986.
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10

Biotinylation and Tetramerization of HLA-E Complexes

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HLA-E-peptide samples requiring biotinylation were subsequently buffered exchanged on Sephadex G-25 PD10 columns (GE Healthcare, UK) into 10mM Tris buffer using commercially available BirA enzyme (Avidity, USA) following the manufacturer’s instructions. Following overnight biotinylation, protein samples were subsequently purified into 20 mM Tris pH 8, 100 mM NaCl buffer or PBS on a HiLoad 16/600 Superdex 75 pg column using an AKTA size-exclusion fast protein liquid chromatography (FPLC) system. Correctly folded β2m-HLA-E*01:03-peptide complexes were subsequently concentrated to 2 mg/ml and snap frozen.
HLA-E*01:03 tetramers were generated via conjugation to various fluorescent labels including Extravidin-PE (Sigma), Streptavidin-bound APC (Biolegend, San Diego) or BV421 (Biolegend, San Diego) at a Molar ratio of 4:1 as previously described12 (link).
Li D., Brackenridge S., Walters L.C., Swanson O., Harlos K., Rozbesky D., Cain D.W., Wiehe K., Scearce R.M., Barr M., Mu Z., Parks R., Quastel M., Edwards R.J., Wang Y., Rountree W., Saunders K.O., Ferrari G., Borrow P., Jones E.Y., Alam S.M., Azoitei M.L., Gillespie G.M., McMichael A.J, & Haynes B.F. (2022). Mouse and human antibodies bind HLA-E-leader peptide complexes and enhance NK cell cytotoxicity. Communications Biology, 5, 271.
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