Lactose Fed-Batch Overexpression of Recombinant Metalloproteins in Escherichia coli BL21(DE3): Process Control Yielding High Levels of Metal-Incorporated, Soluble Protein

Hoffman, Brianna; Broadwater, John A.; Johnson, P. F.; Harper, J. Wade; Fox, Brian G.; Kenealy, William R.

doi:10.1006/prep.1995.1085

Cited by 100 publications

(87 citation statements)

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“…These finding are both consistent with the concept that auto-induction allows a smooth transition of the expression host from an un-induced to induced state under control of natural metabolic processes [28,38,39]. In this case, the need for increased heme biosynthesis was apparently signaled by accumulation of a soluble heme binding protein, but not by expression of an insoluble variant.…”

Section: Auto-inductionsupporting

confidence: 88%

“…Some principles uncovered on the use of auto-induction with the vector platform described here are reported elsewhere [28]. In our previous work on the use of lactose as an inducer of recombinant protein expression, we found that this approach can lead to high-level incorporation of iron into iron-containing proteins [39]. We postulated that this favorable result arose from the slow onset of recombinant protein expression and continued cellular metabolism allowed by lactose-derived induction.…”

Section: Auto-inductionmentioning

confidence: 87%

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A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles

Sobrado

Goren

James

et al. 2008

Protein Expression and Purification

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A specialized vector backbone from the Protein Structure Initiative was used to express full-length human cytochrome b5 as a C-terminal fusion to His8-maltose binding protein in Escherichia coli. The fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of ~18 mg of purified full-length human cytochrome b5 per liter of culture medium was obtained (2.3 mg per]of wet weight bacterial cells). In situ proteolysis of the fusion protein in the presence of chemically defined synthetic liposomes allowed facile spontaneous delivery of the functional peripheral membrane protein into a defined membrane environment without prior exposure to detergents or other lipids. The utility of this approach as a delivery method for production and incorporation of monotopic (peripheral) membrane proteins is discussed.

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Section: Auto-inductionsupporting

confidence: 88%

Section: Auto-inductionmentioning

confidence: 87%

A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles

Sobrado

Goren

James

et al. 2008

Protein Expression and Purification

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“…An 8 M urea extract of bacterial paste derived from 25 liters of a fed-batch fermentation (21) was applied to a 40-cm 3 column of nickel-nitriloacetic acid resin (Ni-NTA, Qiagen, Valencia, CA). Endogenous bacterial proteins were removed by washing with pH 6.0 buffer.…”

Section: Methodsmentioning

confidence: 99%

Cleavage of the Matricellular Protein SPARC by Matrix Metalloproteinase 3 Produces Polypeptides That Influence Angiogenesis

Sage

Reed

Funk

et al. 2003

Journal of Biological Chemistry

150

109

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SPARC, a matricellular protein that affects cellular adhesion and proliferation, is produced in remodeling tissue and in pathologies involving fibrosis and angiogenesis. In this study we have asked whether peptides generated from cleavage of SPARC in the extracellular milieu can regulate angiogenesis. Matrix metalloproteinase (MMP)-3, but not MMP-1 or 9, showed significant activity toward SPARC. Limited digestion of recombinant human (rhu)SPARC with purified catalytic domain of rhuMMP-3 produced three major fragments, which were sequenced after purification by HPLC. Three synthetic peptides (Z-1, Z-2, and Z-3) representing motifs from each fragment were tested in distinct assays of angiogenesis. Peptide Z-1 (3.9 kDa, containing a Cu 2؉ -binding sequence KHGK) exhibited a biphasic effect on [ 3 H]thymidine incorporation by cultured endothelial cells and stimulated vascular growth in the chick chorioallantoic membrane (CAM). In contrast, peptides Z-2 (6.1 kDa, containing Ca 2؉ -binding EF hand-1) and Z-3 (2.2 kDa, containing neither Cu 2؉ -binding motifs nor EF hands), inhibited cell proliferation in a concentration-dependent manner and exhibited no effects on vessel growth in the CAM. Reciprocal results were obtained in a migration assay in native collagen gels: peptide Z-1 was ineffective over a range of concentrations, whereas Z-2 or Z-3 stimulated cell migration. Therefore, proteolysis of SPARC by MMP-3 produced peptides that regulate endothelial cell proliferation and/or migration in vitro in a mutually exclusive manner. One of these peptides containing KHGK also demonstrated a concentration-dependent effect on angiogenesis.

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“…) at 37°C, with a 5-liter min Ϫ1 airflow and an agitation speed maintained at 400 rpm (11). Once the culture had reached an optical density of 0.6 (600 nm; Helios Gamma UV-visible spectrophotometer [Thermo Scientific, Ireland]), the cells were induced with 1 mM IPTG (isopropyl-␤-D-thiogalactopyranoside) and further incubated for 10 to 14 h at 25°C.…”

mentioning

confidence: 99%

FadD from Pseudomonas putida CA-3 Is a True Long-Chain Fatty Acyl Coenzyme A Synthetase That Activates Phenylalkanoic and Alkanoic Acids

2009

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A fatty acyl coenzyme A synthetase (FadD) from Pseudomonas putida CA-3 is capable of activating a wide range of phenylalkanoic and alkanoic acids. It exhibits the highest rates of reaction and catalytic efficiency with long-chain aromatic and aliphatic substrates. FadD exhibits higher k cat and K m values for aromatic substrates than for the aliphatic equivalents (e.g., 15-phenylpentadecanoic acid versus pentadecanoic acid). FadD is inhibited noncompetitively by both acrylic acid and 2-bromooctanoic acid. The deletion of the fadD gene from P. putida CA-3 resulted in no detectable growth or polyhydroxyalkanoate (PHA) accumulation with 10-phenyldecanoic acid, decanoic acid, and longer-chain substrates. The results suggest that FadD is solely responsible for the activation of long-chain phenylalkanoic and alkanoic acids. While the CA-3⌬fadD mutant could grow on medium-chain substrates, a decrease in growth yield and PHA accumulation was observed. The PHA accumulated by CA-3⌬fadD contained a greater proportion of short-chain monomers than did wild-type PHA. Growth of CA-3⌬fadD was unaffected, but PHA accumulation decreased modestly with shorter-chain substrates. The complemented mutant regained 70% to 90% of the growth and PHA-accumulating ability of the wild-type strain depending on the substrate. The expression of an extra copy of fadD in P. putida CA-3 resulted in increased levels of PHA accumulation (up to 1.6-fold) and an increase in the incorporation of longermonomer units into the PHA polymer.Fatty acyl coenzyme A (CoA) synthetases (FACS; fatty acid: CoA ligases; EC 6.2.1.3) are ATP-, CoA-, and Mg 2ϩ -dependent enzymes that activate alkanoic acids to CoA esters for ␤ oxidation ( Fig. 1) (2, 17). FACS are widely distributed in both prokaryotic and eukaryotic organisms and exhibit a broad substrate specificity (34). FadD is a cytoplasmic membrane-associated FACS (7), with sizes ranging from 47 kDa to 62 kDa (2, 14). There is a lack of biochemical information on FadD with a preference for long-chain aromatic and aliphatic substrates. In the current study we purify and characterize for the first time a true long-chain FadD with activity toward both phenylalkanoic and alkanoic acids.It is known that bacteria such as Pseudomonas putida can accumulate the biological polyester polyhydroxyalkanoate (PHA) from aromatic as well as aliphatic alkanoic acids (5, 6, 42, 45). The presence of aromatic monomers in the PHA polymer suggests that a FadD with activity toward aromatic substrates is present in these PHA-accumulating strains. Garcia et al. knocked out an acyl-CoA synthetase in P. putida U with a high homology to long-chain fadD products from Escherichia coli and Pseudomonas fragi (6). Garcia et al. also showed that the mutant was not capable of growth or PHA accumulation with aromatic and aliphatic substrates having between 5 and 10 carbons in their acyl chain, indicating that it is a general and not a long-chain acyl-CoA ligase (6). In a follow-up study, Olivera et al. showed that the fadD mutants reverted to wild-t...

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Lactose Fed-Batch Overexpression of Recombinant Metalloproteins in Escherichia coli BL21(DE3): Process Control Yielding High Levels of Metal-Incorporated, Soluble Protein

Cited by 100 publications

References 0 publications

A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles

A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles

Cleavage of the Matricellular Protein SPARC by Matrix Metalloproteinase 3 Produces Polypeptides That Influence Angiogenesis

FadD from Pseudomonas putida CA-3 Is a True Long-Chain Fatty Acyl Coenzyme A Synthetase That Activates Phenylalkanoic and Alkanoic Acids

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