High-level expression and purification of secreted forms of herpes simplex virus type 1 glycoprotein gD synthesized by baculovirus-infected insect cells

Sisk, William P.; Bradley, Jodi D.; Leipold, Robert J.; Stoltzfus, A M; Leon, Manuel Ponce de; Hilf, Megan; Peng, Chi‐How; Cohen, Gary H.; Eisenberg, Roselyn J.

doi:10.1128/jvi.68.2.766-775.1994

Cited by 107 publications

(77 citation statements)

References 49 publications

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“…pAcVE.02 and pAcVE.03 both encode the honey bee melittin signal peptide (HBM) upstream of the MCS to enhance secretion. 44,57,58 These vankyrin-encoding transfer vectors and their counterparts lacking the P-vank-1 gene (as negative controls) were then used to generate recombinant baculoviruses. Each baculovirus was used to infect Sf9 insect cells, and cell viability was determined up to 10 days post-infection.…”

Section: Resultsmentioning

confidence: 99%

Improving the baculovirus expression vector system with vankyrin‐enhanced technology

Steele

Stone

Franklin

et al. 2017

Biotechnology Progress

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The baculovirus expression vector system (BEVS) is a widely used platform for the production of recombinant eukaryotic proteins. However, the BEVS has limitations in comparison to other higher eukaryotic expression systems. First, the insect cell lines used in the BEVS cannot produce glycoproteins with complex-type N-glycosylation patterns. Second, protein production is limited as cells die and lyse in response to baculovirus infection. To delay cell death and lysis, we transformed several insect cell lines with an expression plasmid harboring a vankyrin gene (P-vank-1), which encodes an anti-apoptotic protein. Specifically, we transformed Sf9 cells, Trichoplusia ni High Five™ cells, and SfSWT-4 cells, which can produce glycoproteins with complex-type N-glycosylation patterns. The latter was included with the aim to increase production of glycoproteins with complex N-glycans, thereby overcoming the two aforementioned limitations of the BEVS. To further increase vankyrin expression levels and further delay cell death, we also modified baculovirus vectors with the P-vank-1 gene. We found that cell lysis was delayed and recombinant glycoprotein yield increased when SfSWT-4 cells were infected with a vankyrin-encoding baculovirus. A synergistic effect in elevated levels of recombinant protein production was observed when vankyrin-expressing cells were combined with a vankyrin-encoding baculovirus. These effects were observed with various model proteins including medically relevant therapeutic proteins. In summary, we found that cell lysis could be delayed and recombinant protein yields could be increased by using cell lines constitutively expressing vankyrin or vankyrin-encoding baculovirus vectors.

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Section: Resultsmentioning

confidence: 99%

Improving the baculovirus expression vector system with vankyrin‐enhanced technology

Steele

Stone

Franklin

et al. 2017

Biotechnology Progress

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“…Production and purification of recombinant baculovirus-produced proteins. The methods for production and DL6 affinity purification of gD1(306t), gD2(306t), and gD2(285t) have been described previously (29,41,48). Production of HveA(200t) (HVEM) and HveC(346t) (nectin-1) by nickel-agarose purification has also been described previously (28,59).…”

Section: Cells and Virusesmentioning

confidence: 99%

Antibody-Induced Conformational Changes in Herpes Simplex Virus Glycoprotein gD Reveal New Targets for Virus Neutralization

Lazear

Whitbeck

Ponce-de-León

et al. 2012

J Virol

134

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As the receptor-binding protein of herpes simplex virus (HSV), gD plays an essential role in virus entry. In its native state, the last 56 amino acids of the ectodomain C terminus (C-term) occlude binding to its receptors, herpesvirus entry mediator (HVEM) and nectin-1. Although it is clear that movement of the C-term must occur to permit receptor binding, we believe that this conformational change is also a key event for triggering later steps leading to fusion. Specifically, gD mutants containing disulfide bonds that constrain the C-term are deficient in their ability to trigger fusion following receptor binding. In this report, we show that two newly made monoclonal antibodies (MAbs), MC2 and MC5, have virus-neutralizing activity but do not block binding of gD to either receptor. In contrast, all previously characterized neutralizing anti-gD MAbs block binding of gD to a receptor(s). Interestingly, instead of blocking receptor binding, MC2 significantly enhances the affinity of gD for both receptors. Several nonneutralizing MAbs (MC4, MC10, and MC14) also enhanced gD-receptor binding. While MC2 and MC5 recognized different epitopes on the core of gD, these nonneutralizing MAbs recognized the gD C-term. Both the neutralizing capacity and rate of neutralization of virus by MC2 are uniquely enhanced when MC2 is combined with MAb MC4, MC10, or MC14. We suggest that MC2 and MC5 prevent gD from performing a function that triggers later steps leading to fusion and that the epitope for MC2 is normally occluded by the C-term of the gD ectodomain.

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“…Plasmid pRE4 (6), which contains the fulllength gD open reading frames of HSV-1 (Patton strain), was used to produce baculovirus recombinants expressing gD truncated at amino acids 285, 275, and 234. DNA fragments were generated by PCR using plasmid pRE4 and the same 5Ј primer as that used for the construction of the recombinant virus bac-gD-1 (306t) (38). The 3Ј primer for gD1(285t) was CGGGAATTCAGTGGTGGTG GTGGTGGTGCGTGCCTACGGGGTCCTCCAAGA.…”

Section: Cells and Virusesmentioning

confidence: 99%

“…The protein products are designated gD1(285t), gD1(275t), and gD1(234t). The strategy used to produce gD1(306t) and gD1(⌬290-299t) has been previously described (33,38).…”

Section: Cells and Virusesmentioning

confidence: 99%

“…Mutations which did not globally alter gD antigenic structure yet resulted in a protein that failed to complement the infectivity of a gD-null virus were grouped into four noncontiguous functional regions, I through IV. To address why these proteins do not function in infection, we used baculovirus vectors to overexpress one truncated, soluble variant from each of the four functional regions (33) as well as wild-type gD from the KOS strain, gD1(306t) (38). Three of the mutants showed diminished or no ability to block infection compared to wild-type gD.…”

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confidence: 99%

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Functional Region IV of Glycoprotein D from Herpes Simplex Virus Modulates Glycoprotein Binding to the Herpesvirus Entry Mediator

Rux

Willis

Nicola

et al. 1998

J Virol

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Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry and has four functional regions (I to IV) important for this process. We previously showed that a truncated form of a functional region IV variant, gD1(⌬290-299t), had an enhanced ability to block virus entry and to bind to the herpesvirus entry mediator (HveAt; formerly HVEMt), a cellular receptor for HSV. To explore this phenotype further, we examined other forms of gD, especially ones with mutations in region IV. Variant proteins with deletions of amino acids between 277 and 300 (region IV), as well as truncated forms lacking C-terminal residues up to amino acid 275 of gD, were able to block HSV entry into Vero cells 1 to 2 logs better than wild-type gD1(306t). In contrast, gD truncated at residue 234 did not block virus entry into Vero cells. Using optical biosensor technology, we recently showed that gD1(⌬290-299t) had a 100-fold-higher affinity for HveAt than gD1(306t) (3.3 ؋ 10 ؊8 M versus 3.2 ؋ 10 ؊6 M). Here we found that the affinities of other region IV variants for HveAt were similar to that of gD1(⌬290-299t). Thus, the affinity data follow the same hierarchy as the blocking data. In each case, the higher affinity was due primarily to a faster k on rather than to a slower k off . Therefore, once the gDt-HveAt complex formed, its stability was unaffected by mutations in or near region IV. gD truncated at residue 234 bound to HveAt with a lower affinity (2.0 ؋ 10 ؊5 M) than did gD1(306t) due to a more rapid k off . These data suggest that residues between 234 and 275 are important for maintaining stability of the gDt-HveAt complex and that functional region IV is important for modulating the binding of gD to HveA. The binding properties

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High-level expression and purification of secreted forms of herpes simplex virus type 1 glycoprotein gD synthesized by baculovirus-infected insect cells

Cited by 107 publications

References 49 publications

Improving the baculovirus expression vector system with vankyrin‐enhanced technology

Improving the baculovirus expression vector system with vankyrin‐enhanced technology

Antibody-Induced Conformational Changes in Herpes Simplex Virus Glycoprotein gD Reveal New Targets for Virus Neutralization

Functional Region IV of Glycoprotein D from Herpes Simplex Virus Modulates Glycoprotein Binding to the Herpesvirus Entry Mediator

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