Molecular Interaction Between the Strep-tag Affinity Peptide and its Cognate Target, Streptavidin

Schmidt, Torben; Koepke, Jürgen; Frank, Ronald; Skerra, Arne

doi:10.1006/jmbi.1996.0061

Cited by 305 publications

(252 citation statements)

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“…To verify these assumptions, we established an in vitro system for the conversion of apo-ACP His into AMP-ACP His with purified proteins. For this purpose, the expression plasmid pET224-citXstrep was constructed, encoding a CitX derivative with the Strep-tag II peptide (32). CitX Strep (193 amino acid residues, 21.5 kDa) contained 10 additional residues (TSWSHPQFEK) at the C-terminus, which allowed purification by StrepTactin affinity chromatography (35).…”

Section: Identification Of the Genes Required For The Biosynthesis Ofmentioning

confidence: 99%

Biosynthesis of the Prosthetic Group of Citrate Lyase

2000

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Citrate lyase (EC 4.1.3.6) catalyzes the cleavage of citrate to acetate and oxaloacetate and is composed of three subunits (R, , and γ). The γ-subunit serves as an acyl carrier protein (ACP) and contains the prosthetic group 2′-(5′′-phosphoribosyl)-3′-dephospho-CoA, which is attached via a phosphodiester linkage to serine-14 in the enzyme from Klebsiella pneumoniae. In this work, we demonstrate by genetic and biochemical studies with citrate lyase of Escherichia coli and K. pneumoniae that the conversion of apo-ACP into holo-ACP is dependent on the two proteins, CitX (20 kDa) and CitG (33 kDa). In the absence of CitX, only apo-ACP was synthesized in vivo, whereas in the absence of CitG, an adenylylated ACP was produced, with the AMP residue attached to serine-14. The adenylyltransferase activity of CitX could be verified in vitro with purified CitX and apo-ACP plus ATP as substrates. Besides ATP, CTP, GTP, and UTP also served as nucleotidyl donors in vitro, showing that CitX functions as a nucleotidyltransferase. The conversion of apo-ACP into holo-ACP was achieved in vitro by incubation of apo-ACP with CitX, CitG, ATP, and dephospho-CoA. ATP could not be substituted with GTP, CTP, UTP, ADP, or AMP. In the absence of CitG or dephospho-CoA, AMP-ACP was formed. Remarkably, it was not possible to further convert AMP-ACP to holo-ACP by subsequent incubation with CitG and dephospho-CoA. This demonstrates that AMP-ACP is not an intermediate during the conversion of apo-into holo-ACP, but results from a side activity of CitX that becomes effective in the absence of its natural substrate. Our results indicate that holo-ACP formation proceeds as follows. First, a prosthetic group precursor [presumably 2′-(5′′-triphosphoribosyl)-3′-dephospho-CoA] is formed from ATP and dephospho-CoA in a reaction catalyzed by CitG. Second, holo-ACP is formed from apo-ACP and the prosthetic group precursor in a reaction catalyzed by CitX.Citrate lyase (EC 4.1.3.6) catalyzes the Mg 2+ -dependent cleavage of citrate to acetate and oxaloacetate ( Figure 1A). This reaction represents the initial step of all known bacterial citrate fermentation pathways (1-4). Citrate lyase has been isolated from numerous bacterial species, while the biochemical characterization was performed mainly with the enzyme from Klebsiella pneumoniae (formerly Klebsiella aerogenes). It is a complex of 550 kDa, formed by six copies of each of the three different subunits [R (55 kDa), (32 kDa), and γ (11 kDa)] (5, 6). The catalytic activity resides on the R-and -subunits, whereas the γ-subunit serves as an acyl carrier protein (ACP) 1 (7) containing 2′-(5′′-phosphoribosyl)-3′-dephospho-CoA as a prosthetic group ( Figure 1B). This group is attached by its ribose 5′-phosphate moiety via a phosphodiester linkage to serine-14 of the ACP (8-12). As a prerequisite for catalytic activity, the thiol group of the enzyme-bound CoA derivative needs to be acetylated, resulting in the formation of an acetyl thioester (7,13).

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Section: Identification Of the Genes Required For The Biosynthesis Ofmentioning

confidence: 99%

Biosynthesis of the Prosthetic Group of Citrate Lyase

2000

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“…Codon-optimized sequences for a polyhistidine (His 6 -) affinity tag (Porath et al 1975) and a streptavidin (StrepII-) affinity tag (Schmidt et al 1996) were cloned into the expression vector for the synthesis of N-terminal (Fig. 5B) or C-terminally (Fig.…”

Section: Rna Isolation and Northern Detectionmentioning

confidence: 99%

A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger

Roth

Dersch

2009

Appl Microbiol Biotechnol

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“…Each streptavidin monomer binds one biotin molecule with femtomolar affinity, one of the strongest non-covalent interactions observed in nature [8]. Over the past 15 years several groups have developed peptide binders to the tetrameric streptavidin protein [9][10][11][12][13] and a variety of protein engineering approaches have been applied to tailor affinity for specific applications [14][15][16]. This provides a simple and clean system for protein purification as a streptavidin binding peptide can be selectively eluted from a streptavidin affinity column using appropriate concentrations of biotin.…”

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confidence: 99%

“…The Strep-tag, although useful for the one-step purification of recombinant proteins [17], could only be used in the C-terminus of the protein, as a free carboxy-terminus is required for binding. This deficit motivated the development of the StrepII-tag, which is similar in length and can be included anywhere within the protein sequence [10]. Both these tags contain the conserved HPQ amino acid motif, which is found in many streptavidin binding peptides and is chiefly involved in mediating the interaction with streptavidin.…”

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confidence: 99%

“…Both these tags contain the conserved HPQ amino acid motif, which is found in many streptavidin binding peptides and is chiefly involved in mediating the interaction with streptavidin. Although the shortness of the Strep-tags makes them very attractive as biochemical tools, both bind streptavidin with relatively low affinity (37µM and 72µM, respectively [10]). This poses a problem in cases where higher affinity is required e.g.…”

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