scholarly journals Adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii. Active holoenzyme produced from Escherichia coli

1990 ◽  
Vol 269 (2) ◽  
pp. 293-298 ◽  
Author(s):  
N McKie ◽  
N H Keep ◽  
M L Patchett ◽  
P F Leadlay
Keyword(s):  
Escherichia Coli ◽  
Cellular Protein ◽  
Site Directed Mutagenesis ◽  
Soluble Form ◽  
Beta Subunits ◽  
Structural Genes ◽  
Positive Bacterium ◽  
Total Cellular Protein ◽  
Propionibacterium Shermanii ◽  
Phage T7

The linked structural genes coding for both subunits of adenosylcobalamin-dependent methylmalonyl-CoA mutase from the Gram-positive bacterium Propionibacterium shermanii have been altered by site-directed mutagenesis and placed under the control of an inducible phage-T7-specific plasmid promoter in Escherichia coli. Conditions have been found under which both alpha- and beta-subunits are produced in soluble form, in near 1:1 ratio, and assemble to form apo-mutase totalling about 5% of the total cellular protein. Methylmalonyl-CoA mutase purified from these cells could be readily converted into the holoenzyme by addition of adenosylcobalamin. The active holoenzyme apparently crystallizes in the same space group as an inactive corrinoid-containing form of the enzyme obtained previously.

scholarly journals Biosynthesis of Cyanobacterial Phycobiliproteins in Escherichia coli: Chromophorylation Efficiency and Specificity of All Bilin Lyases from Synechococcus sp. Strain PCC 7002

2010 ◽  
Vol 76 (9) ◽  
pp. 2729-2739 ◽  
Author(s):  
Avijit Biswas ◽  
Yasmin M. Vasquez ◽  
Tierna M. Dragomani ◽  
Monica L. Kronfel ◽  
Shervonda R. Williams ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biosynthetic Pathway ◽  
Expression System ◽  
Water Soluble ◽  
Soluble Form ◽  
Beta Subunits ◽  
Lyase Activity ◽  
Synechococcus Sp ◽  
Tetrapyrrole Chromophores

ABSTRACT Phycobiliproteins are water-soluble, light-harvesting proteins that are highly fluorescent due to linear tetrapyrrole chromophores, which makes them valuable as probes. Enzymes called bilin lyases usually attach these bilin chromophores to specific cysteine residues within the alpha and beta subunits via thioether linkages. A multiplasmid coexpression system was used to recreate the biosynthetic pathway for phycobiliproteins from the cyanobacterium Synechococcus sp. strain PCC 7002 in Escherichia coli. This system efficiently produced chromophorylated allophycocyanin (ApcA/ApcB) and α-phycocyanin with holoprotein yields ranging from 3 to 12 mg liter−1 of culture. This heterologous expression system was used to demonstrate that the CpcS-I and CpcU proteins are both required to attach phycocyanobilin (PCB) to allophycocyanin subunits ApcD (αAP-B) and ApcF (β18). The N-terminal, allophycocyanin-like domain of ApcE (LCM 99) was produced in soluble form and was shown to have intrinsic bilin lyase activity. Lastly, this in vivo system was used to evaluate the efficiency of the bilin lyases for production of β-phycocyanin.

scholarly journals An Exocellular Protein from the Oil-Degrading Microbe Acinetobacter venetianus RAG-1 Enhances the Emulsifying Activity of the Polymeric Bioemulsifier Emulsan

2003 ◽  
Vol 69 (5) ◽  
pp. 2608-2615 ◽  
Author(s):  
Horacio Bach ◽  
Yevgeny Berdichevsky ◽  
David Gutnick
Keyword(s):  
Inclusion Bodies ◽  
Acinetobacter Calcoaceticus ◽  
Site Directed Mutagenesis ◽  
Soluble Form ◽  
Emulsifying Activity ◽  
T7 Promoter ◽  
Cell Extracts ◽  
Phage T7 ◽  
Oil Water ◽  
Rag 1

ABSTRACT The oil-degrading microorganism Acinetobacter venetianus RAG-1 produces an extracellular polyanionic, heteropolysaccharide bioemulsifier termed emulsan. Emulsan forms and stabilizes oil-water emulsions with a variety of hydrophobic substrates. Removal of the protein fraction yields a product, apoemulsan, which exhibits much lower emulsifying activity on hydrophobic substrates such as n-hexadecane. One of the key proteins associated with the emulsan complex is a cell surface esterase. The esterase (molecular mass, 34.5 kDa) was cloned and overexpressed in Escherichia coli BL21(DE3) behind the phage T7 promoter with the His tag system. After overexpression, about 80 to 90% of the protein was found in inclusion bodies. The overexpressed esterase was recovered from the inclusion bodies by solubilization with deoxycholate and, after slow dialysis, was purified by metal chelation affinity chromatography. Mixtures containing apoemulsan and either the catalytically active soluble form of the recombinant esterase isolated from cell extracts or the solubilized inactive form of the enzyme recovered from the inclusion bodies formed stable oil-water emulsions with very hydrophobic substrates such as hexadecane under conditions in which emulsan itself was ineffective. Similarly, a series of esterase-defective mutants were generated by site-directed mutagenesis, cloned, and overexpressed in E. coli. Mutant proteins defective in catalytic activity as well as others apparently affected in protein conformation were also active in enhancing the apoemulsan-mediated emulsifying activity. Other proteins, including a His-tagged overexpressed esterase from the related organism Acinetobacter calcoaceticus BD4, showed no enhancement.

scholarly journals Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase

1992 ◽  
Vol 267 (32) ◽  
pp. 22830-22836 ◽  
Author(s):  
K Ostanin ◽  
E.H. Harms ◽  
P.E. Stevis ◽  
R Kuciel ◽  
M.M. Zhou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acid Phosphatase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit.

1994 ◽  
Vol 180 (6) ◽  
pp. 2147-2153 ◽  
Author(s):  
M Pizza ◽  
M R Fontana ◽  
M M Giuliani ◽  
M Domenighini ◽  
C Magagnoli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Neutralizing Antibodies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
E Coli ◽  
Heat Labile ◽  
A Subunit ◽  
Adp Ribosyltransferase ◽  
Derivatives Of

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.

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