Release of Proteins and Peptides from Fusion Proteins Using a Recombinant Plant Virus Proteinase

Parks, T. Dawn; Leuther, Kerstin K.; Howard, E.D.; Johnston, Sean; Dougherty, William G.

doi:10.1006/abio.1994.1060

Cited by 280 publications

(168 citation statements)

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“…ALP and CLP36 fragments were cloned in a modified pET24d vector (Novagen, Merck Biosciences, Schwalbach, Germany). All inserts in the modified pET24d vector contained an N-terminal His 6 tag followed by a tobacco etch virus protease recognition site (27) and three extra residues (ENLYFQ2GAMG, 2 denotes the tobacco etch virus cleavage site). Full human ␣-actinin 2A (M86406) (28) in modified pET8c (Novagen) vector (described in Djinovic-Carugo et al (3)) was used to generate shorted fragments of ␣-actinin 2 to the pET8c or to the pET24d vector.…”

Section: Methodsmentioning

confidence: 99%

The ZASP-like Motif in Actinin-associated LIM Protein Is Required for Interaction with the α-Actinin Rod and for Targeting to the Muscle Z-line

Klaavuniemi

Kelloniemi

Ylänne

2004

Journal of Biological Chemistry

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The Z-line is a specialized structure connecting adjacent sarcomeres in muscle cells. ␣-Actinin cross-links actin filaments in the Z-line. Several PDZ-LIM domain proteins localize to the Z-line and interact with ␣-actinin. Actinin-associated LIM protein (ALP), C-terminal LIM domain protein (CLP36), and Z band alternatively spliced PDZ-containing protein (ZASP) have a conserved region named the ZASP-like motif (ZM) between PDZ and LIM domains. To study the interactions and function of ALP we used purified recombinant proteins in surface plasmon resonance measurements. We show that ALP and ␣-actinin 2 have two interaction sites. The ZM motif was required for the interaction of ALP internal region with the ␣-actinin rod and for targeting of ALP to the Z-line. The PDZ domain of ALP bound to the C terminus of ␣-actinin. This is the first indication that the ZM motif would have a direct role in a proteinprotein interaction. These results suggest that the two interaction sites of ALP would stabilize certain conformations of ␣-actinin 2 that would strengthen the Z-line integrity.The muscle Z-line is a highly specialized structure between adjacent sarcomeres in muscle fibers that maintain the organization of the contractile machinery (for review, see Ref. 1). ␣-Actinin is one of the major components at the Z-line. The ␣-actinin polypeptide is composed of an N-terminal actin binding domain, four spectrin repeats that form the central rod region (2, 3), and two pairs of C-terminal EF-hands (4). ␣-Actinin forms an antiparallel homodimer. ␣-Actinin cross-links actin filaments from opposite sarcomeres to the Z-line and, therefore, has a major mechanical function in keeping the sarcomeres together.Several PDZ-LIM proteins have been detected in the muscle Z-line and shown to interact with ␣-actinin (5-12). PDZ domain is a widely expressed protein-protein interaction domain (for review, see Ref. 13). PDZ-LIM proteins form one subgroup of PDZ proteins (13) and are regarded as mediators between cytoskeletal structures and signaling cascades.PDZ-LIM proteins can be divided in two subclasses; they either contain one or three LIM domains. Actinin-associated LIM protein (ALP) 1 (5, 10), C-terminal LIM domain protein (CLP36) (14) (also called hCLIM1 (15) or Elfin (16)), Reversion-induced LIM protein (RIL) (17), and Mystique (Uniprot accession number Q7Z584) belong to the first class, which has one N-terminal PDZ domain and one C-terminal LIM domain. ALP is expressed in muscle (5, 10), whereas CLP36 and RIL are mainly expressed in nonmuscle tissues (11,18,19). CLP36 shows also high expression levels in muscle (7,15,16). Enigma, Enigma homology protein, and ZASP/Cypher/Oracle form the second class, with one N-terminal PDZ domain and three C-terminal LIM domains. They all are expressed mainly in muscle (6, 9, 20 -22). An interesting feature of these seven PDZ-LIM proteins is that ALP, CLP36, and ZASP contain a conserved region, named ZASP-like motif (ZM) (SMART prediction (23) accession number SM 00735, Interpro 006643), in the internal r...

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Section: Methodsmentioning

confidence: 99%

The ZASP-like Motif in Actinin-associated LIM Protein Is Required for Interaction with the α-Actinin Rod and for Targeting to the Muscle Z-line

Klaavuniemi

Kelloniemi

Ylänne

2004

Journal of Biological Chemistry

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“…Furthermore, the forward primer 5 0 -CATTCCATGGGTTCCGACGACCAGCTGGTG (NcoI restriction site in bold) and reverse primer 5 0 -TAAGGTACCTCAGCGGCC-GGAGTTGGCGAGTTTCTC were used to introduce an extra arginine residue (in bold) at the C-terminal end of the protein construct (construct MafB 211-305,C298S,R306 ; all primers were synthesized by MWG-Biotech). The amplified PCR product was subcloned into a pETM-11 expression vector which encodes an N-terminal 6ÂHis tag and a TEV cleavage site for subsequent affinity-tag removal by TEV protease (Parks et al, 1994). Optimal recombinant protein expression was obtained using Escherichia coli strain BL21(DE3) Codon Plus RIL (Stratagene).…”

Section: Cloning and Expressionmentioning

confidence: 99%

Expression, purification, crystallization and preliminary crystallographic analysis of the mouse transcription factor MafB in complex with its DNA-recognition motif Cmare

Textor¹,

Wilmanns²,

Holton³

2007

Acta Cryst Sect F

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The MafB transcription factor (residues 211-305) has been overexpressed in and purified from Escherichia coli. A protein-DNA complex between the MafB homodimer and the 21 bp Maf-recognition sequence known as Cmare has been successfully reconstituted in vitro and subsequently crystallized. The diffraction properties of the protein-DNA complex crystals were improved using a combination of protein-construct boundary optimization and targeted mutagenesis to promote crystal lattice stability. Both native and mercury-derivatized crystals have been prepared using these optimized conditions. The crystals belong to space group P4 1 2 1 2 or P4 3 2 1 2, with unit-cell parameters a = b = 94.8, c = 197.9 Å . An anomalous difference Patterson map computed using data collected from crystals grown in the presence of HgCl 2 reveals four peaks. This corresponds to two copies of the protein-DNA complex in the asymmetric unit, with a solvent content of 62% and a Matthews coefficient of 3.22 Å 3 Da À1 .

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“…One method of tag removal is through the use of the tobacco etch virus (TEV) protease. TEV protease has become one of the proteases of choice for cleaving fusion proteins due to its high degree of specificity, its resistance to many protease inhibitors used in protein purification, and the ease of separation of both the protease and affinity tag from the protein of interest (Parks et al, 1994). Additionally, improvement to the ability to purify large amounts of TEV protease (Blommel & Fox, 2007;Lucast et al, 2001;van den Berg et al, 2006) can make the TEV protease a relatively inexpensive option for cleavage of fusion proteins when compared to other commercially available options.…”

Section: Introductionmentioning

confidence: 99%

Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli

Rocco

Dennison

Klenchin

et al. 2008

Plasmid

119

121

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We describe the construction and use of two sets of vectors for the over-expression and purification of protein from E. coli. The set of pTEV plasmids (pTEV3, 4, 5) directs the synthesis of a recombinant protein with a N-terminal hexahistidine (His 6 ) tag that is removable by the tobacco etch virus (TEV) protease. The set of pKLD plasmids (pKLD66, 116) directs the synthesis of a recombinant protein that contains a N-terminal His 6 and maltose-binding protein tags in tandem, which can also be removed with TEV protease. The usefulness of these plasmids is illustrated by the rapid, high-yield purification of the 2-methylcitrate dehydratase (PrpD) protein of Salmonella enterica, and the 2-methylaconitate isomerase (PrpF) protein of Shewanella oneidensis, two enzymes involved in the catabolism of propionate to pyruvate via the 2-methylcitric acid cycle.

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Release of Proteins and Peptides from Fusion Proteins Using a Recombinant Plant Virus Proteinase

Cited by 280 publications

References 0 publications

The ZASP-like Motif in Actinin-associated LIM Protein Is Required for Interaction with the α-Actinin Rod and for Targeting to the Muscle Z-line

The ZASP-like Motif in Actinin-associated LIM Protein Is Required for Interaction with the α-Actinin Rod and for Targeting to the Muscle Z-line

Expression, purification, crystallization and preliminary crystallographic analysis of the mouse transcription factor MafB in complex with its DNA-recognition motif Cmare

Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli

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