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A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae).

Scavuzzo-Duggan TR, Chaves AM, Roberts AW - Appl Plant Sci (2015)

Bottom Line: A method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens.The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins.Compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Rhode Island, 120 Flagg Road, Kingston, Rhode Island 02881 USA.

ABSTRACT

Premise of the study: A method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens.

Methods and results: A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Western blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins.

Conclusions: Compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.

No MeSH data available.


Related in: MedlinePlus

PCR products separated on a 1% agarose gel (100 V, 25 min). Concentrations of F1 and F2 were both estimated at ∼240 ng/μL. However, F1 is ∼2500 bp and F2 is ∼1000 bp. Thus, 2 μL of PCR reaction F1 was combined with 1.33 μL of PCR reaction F2 for the PCR fusion reaction.
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figa2: PCR products separated on a 1% agarose gel (100 V, 25 min). Concentrations of F1 and F2 were both estimated at ∼240 ng/μL. However, F1 is ∼2500 bp and F2 is ∼1000 bp. Thus, 2 μL of PCR reaction F1 was combined with 1.33 μL of PCR reaction F2 for the PCR fusion reaction.

Mentions: Expression vectors carrying a wild-type (positive control) or a mutated (test) PpCESA5 coding sequence fused at the N-terminus to a triple hemagglutinin (3XHA) epitope tag were constructed using PCR fusion and MultiSite Gateway cloning (Atanassov et al., 2009). To create Gateway entry clones carrying point mutations, gene fragments were amplified from wild-type PpCESA5 cDNA clone pdp24095 (RIKEN BioResource Center, http://www.brc.riken.jp/lab/epd/Eng/) using primer pairs that added an att site at one end and introduced a mutation at the other (Appendix 1; Appendix 2, Fig. A1, A2). The fragments were fused in a single overlap extension reaction (Appendix 2) and cloned into pDONR 221 P5-P2 according to the manufacturer’s instructions (Life Technologies, Grand Island, New York, USA). CESA5 entry clones were sequence verified using primers listed in Appendix 3. To create the 3XHA entry clone, two oligonucleotides (Appendix 1) were fused, cloned into pDONR 221 P1-P5r (Life Technologies), and sequence verified using primer M13 Forward (−20) (Life Technologies). Each CESA5 entry clone was transferred along with a 3XHA entry clone to the pTHAct1Gate destination vector using LR Clonase II Plus as described by the manufacturer (Life Technologies). The pTHAct1Gate vector contains an Act1 promoter from rice, which drives constitutive expression in P. patens, and sequences that target the vector to the P. patens 108 locus, which can be disrupted without producing a phenotype (Perroud and Quatrano, 2006).


A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae).

Scavuzzo-Duggan TR, Chaves AM, Roberts AW - Appl Plant Sci (2015)

PCR products separated on a 1% agarose gel (100 V, 25 min). Concentrations of F1 and F2 were both estimated at ∼240 ng/μL. However, F1 is ∼2500 bp and F2 is ∼1000 bp. Thus, 2 μL of PCR reaction F1 was combined with 1.33 μL of PCR reaction F2 for the PCR fusion reaction.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4504723&req=5

figa2: PCR products separated on a 1% agarose gel (100 V, 25 min). Concentrations of F1 and F2 were both estimated at ∼240 ng/μL. However, F1 is ∼2500 bp and F2 is ∼1000 bp. Thus, 2 μL of PCR reaction F1 was combined with 1.33 μL of PCR reaction F2 for the PCR fusion reaction.
Mentions: Expression vectors carrying a wild-type (positive control) or a mutated (test) PpCESA5 coding sequence fused at the N-terminus to a triple hemagglutinin (3XHA) epitope tag were constructed using PCR fusion and MultiSite Gateway cloning (Atanassov et al., 2009). To create Gateway entry clones carrying point mutations, gene fragments were amplified from wild-type PpCESA5 cDNA clone pdp24095 (RIKEN BioResource Center, http://www.brc.riken.jp/lab/epd/Eng/) using primer pairs that added an att site at one end and introduced a mutation at the other (Appendix 1; Appendix 2, Fig. A1, A2). The fragments were fused in a single overlap extension reaction (Appendix 2) and cloned into pDONR 221 P5-P2 according to the manufacturer’s instructions (Life Technologies, Grand Island, New York, USA). CESA5 entry clones were sequence verified using primers listed in Appendix 3. To create the 3XHA entry clone, two oligonucleotides (Appendix 1) were fused, cloned into pDONR 221 P1-P5r (Life Technologies), and sequence verified using primer M13 Forward (−20) (Life Technologies). Each CESA5 entry clone was transferred along with a 3XHA entry clone to the pTHAct1Gate destination vector using LR Clonase II Plus as described by the manufacturer (Life Technologies). The pTHAct1Gate vector contains an Act1 promoter from rice, which drives constitutive expression in P. patens, and sequences that target the vector to the P. patens 108 locus, which can be disrupted without producing a phenotype (Perroud and Quatrano, 2006).

Bottom Line: A method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens.The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins.Compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Rhode Island, 120 Flagg Road, Kingston, Rhode Island 02881 USA.

ABSTRACT

Premise of the study: A method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens.

Methods and results: A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Western blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins.

Conclusions: Compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.

No MeSH data available.


Related in: MedlinePlus