Preventing Proteolytic Artifacts in the Baculovirus Expression System

Hom, Louis G.; Volkman, Loy E.

doi:10.2144/98251bm01

Cited by 31 publications

(13 citation statements)

References 3 publications

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“…. Note that the 45-kDa band detected by 7E11 was likely due to the proteolytic degradation in the crude insect cell lysates or an artifact in SDS-PAGE sample preparation (26). Taken together, Western blot analyses demonstrated that rPSMA was correctly expressed in the insect cells and also implied that the addition of six histidine amino acid residues to the amino terminus of PSMA did not mask or interfere with the epitope recognized by MoAb 7E11.…”

Section: Generation and Identification Of Rpsma Baculovirusmentioning

confidence: 87%

Generation of a Baculovirus Recombinant Prostate-Specific Membrane Antigen and Its Use in the Development of a Novel Protein Biochip Quantitative Immunoassay

Xiao

Jiang

Beckett

et al. 2000

Protein Expression and Purification

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Section: Generation and Identification Of Rpsma Baculovirusmentioning

confidence: 87%

Generation of a Baculovirus Recombinant Prostate-Specific Membrane Antigen and Its Use in the Development of a Novel Protein Biochip Quantitative Immunoassay

Xiao

Jiang

Beckett

et al. 2000

Protein Expression and Purification

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“…Perhaps the close association of proV-CATH in a complex with CHIA is less prone to premature proteolytic maturation to V-CATH (by either cellular or extracellular proteases) (16,21,22) due to CHIA blocking its prodomain cleavage site, which would not be possible for the chiA-deficient ⌬CH/CA virus.…”

Section: Discussionmentioning

confidence: 99%

“…ProV-CATH expressed by (lacZ-inactivated) chiA-deficient viruses, in addition to being reported as insoluble, was also incompetent to induction with chaotropic agents such as acids or SDS into autoproteolytic maturation into V-CATH (1,16). We wanted to see if the proV-CATH produced in our ⌬chiA bacmid system could be induced with an acidic buffer to mature autoproteolytically into V-CATH.…”

mentioning

confidence: 99%

Role of Interactions between Autographa californica Multiple Nucleopolyhedrovirus Procathepsin and Chitinase Chitin-Binding or Active-Site Domains in Viral Cathepsin Processing

Hodgson

Arif

Krell

2013

J Virol

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The binding of Autographa californica multiple nucleopolyhedrovirus chitinase (CHIA) to viral cathepsin protease progenitor (proV-CATH) governs cellular/endoplasmic reticulum (ER) coretention of CHIA and proV-CATH, thus coordinating simultaneous cellular release of both host tissue-degrading enzymes upon host cell death. CHIA is a proposed proV-CATH folding chaperone because insertional inactivation of chiA causes production of proV-CATH aggregates that are incompetent for proteolytic maturation into active V-CATH enzyme. We wanted to determine whether the N-terminal chitin-binding domain (CBD, 149 residues) and C-terminal CHIA active-site domain (ASD, 402 residues) of CHIA bind to proV-CATH independently of one another and whether either domain is dispensable for CHIA's putative proV-CATH folding chaperone activity. We demonstrate that N-terminally green fluorescent protein (GFP)-fused CHIA, ASD, and CBD each colocalize with proV-CATH-RFP in ER-like patterns and that both ASD and CBD independently associate with proV-CATH in vivo using bimolecular fluorescence complementation (BiFC) and in vitro using reciprocal nickel-histidine pulldown assays. Altogether, the data from colocalization, BiFC, and reciprocal copurification analyses suggest specific and independent interactions between proV-CATH and both domains of CHIA. These data also demonstrate that either CHIA domain is dispensable for normal proV-CATH processing. Furthermore, in contrast to prior evidence suggesting that a lack of chiA expression causes proV-CATH to become aggregated, insoluble, and unable to mature into V-CATH, a chiA deletion bacmid virus we engineered to express just v-cath produced soluble proV-CATH that was prematurely secreted from cells and proteolytically matured into active V-CATH enzyme.

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“…In addition, cell lysis may leak significant amounts of the engineered proteins into the medium, which are difficult to recover. In order to overcome protein degradation in BEVSs, efforts have been made to inhibit protease activity, including addition of inhibitor cocktails to the culture medium and elimination of viral protease from the viral genome [7,11,12]. However, protease inhibitors may impair cell growth when included in culture medium, and they are expensive, particularly in large-scale expression systems.…”

Section: Introductionmentioning

confidence: 99%

Enhancement of correct protein folding in vivo by a non-lytic baculovirus

Lee

et al. 2004

Biochemical Journal

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The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7 % cell lysis, whereas L-BEVS showed 60 % lysis of cells. The quality of protein expressed in both N-and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP-LUC-ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRETbased in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.

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Preventing Proteolytic Artifacts in the Baculovirus Expression System

Cited by 31 publications

References 3 publications

Generation of a Baculovirus Recombinant Prostate-Specific Membrane Antigen and Its Use in the Development of a Novel Protein Biochip Quantitative Immunoassay

Generation of a Baculovirus Recombinant Prostate-Specific Membrane Antigen and Its Use in the Development of a Novel Protein Biochip Quantitative Immunoassay

Role of Interactions between Autographa californica Multiple Nucleopolyhedrovirus Procathepsin and Chitinase Chitin-Binding or Active-Site Domains in Viral Cathepsin Processing

Enhancement of correct protein folding in vivo by a non-lytic baculovirus

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