Abstract:
:Recombinant protein expression from lac derived promoters by the autoinduction regime is based on diauxic growth of Escherichia coli on glucose and lactose. Glycerol is used as a supporting carbon source during the lactose-induced expression. While this glycerol-based formulation usually provides high cell densities, successful protein expression by autoinduction is often very dependent on correct aeration level. This complicates the reproducibility and scalability of the cultures. In this study we investigate the use of an alternative autoinduction formulation, in which the supporting carbon source is provided by fed-batch-like slow glucose feed from a biocatalytically degraded polysaccharide. The glucose feed as supporting carbon source allowed for high level of autoinduced target protein expression from T7lac promoter in E. coli BL21(DE3) and from T5lac promoter in E. coli K-12 RB791(lacI(q)) with lactose concentrations of 0.5-2gl(-1). Cell densities and protein yields per culture volume were similar to or higher than in the glycerol-based ZYM-5052 medium. In the glycerol-based medium, protein production was adversely influenced by high aeration level, resulting in 75-90% reduction in protein yield per cell compared to more moderately aerated conditions. The glucose fed-batch medium attenuated this oxygen-sensitivity and provided robust high-yield expression also under high aeration rates. It is concluded that the slow glucose feed as supporting carbon source mitigates aeration-related scale differences in autoinduced protein expression, and combined with the benefit of high product yields this makes the fed-batch autoinduction medium ideal for high-throughput screening and scale-up of the production process.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Ukkonen K,Mayer S,Vasala A,Neubauer Pdoi
10.1016/j.pep.2013.07.016subject
Has Abstractpub_date
2013-10-01 00:00:00pages
147-54issue
2eissn
1046-5928issn
1096-0279pii
S1046-5928(13)00142-3journal_volume
91pub_type
杂志文章abstract::Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin therapy. In our previous study, a modified PE38KDEL, denoted PE38KDELKQK, was engineered to eliminate VLS. The PE38KDELKQK-based immunotoxin has been proved to retain potent anti-tumor activity but with a remarkable attenuation in VLS. In ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.09.009
更新日期:2008-03-01 00:00:00
abstract::We previously reported the expression and purification of recombinant p68 RNA helicase in a bacterial expression system. The recombinant p68 is an RNA-dependent ATPase and ATP-dependent RNA helicase. In the process of characterizing the ATPase and RNA unwinding activities of the recombinant p68, we observed that the b...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2004.02.004
更新日期:2004-06-01 00:00:00
abstract::The B-subunits of replicative DNA polymerases belong to the superfamily of calcineurin-like phosphoesterases and are conserved from Archaea to humans. Recently we and others have shown that the B-subunit (DP1) of the archaeal family D DNA polymerase is responsible for proofreading 3'-5' exonuclease activity. The simil...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2005.05.002
更新日期:2005-09-01 00:00:00
abstract::Endostatin, a carboxy-terminal fragment of collagen XVIII, has been shown to act as an anti-angiogenic agent that specifically inhibits proliferation of endothelial cells and growth of various primary tumors. Here, we describe the expression by Chinese hamster ovary (CHO) cells of murine endostatin and of a tagged-fus...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2004.01.003
更新日期:2004-05-01 00:00:00
abstract::An unknown d-tagatose 3-epimerase (DTE) containing a IoIE domain was identified and cloned from Escherichia coli. This gene was subcloned into the prokaryotic expression vector pET-15b, and induced by IPTG in E. coli BL21 expression system. Through His-select gel column purification and fast-protein liquid chromatogra...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2015.06.015
更新日期:2015-10-01 00:00:00
abstract::Structural studies of human peptide hormone somatostatin 14 (SS14) require high amounts of isotopically labelled SS14 to be produced. Here we report a method for effective production of isotopically labelled SS14. SS14 was expressed as a fusion protein with thioredoxin in Escherichia coli. Co-expression of a longer po...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2014.03.011
更新日期:2014-07-01 00:00:00
abstract::Human cystatin C (HCC), encoded by cystatin 3 gene, is a 13.3kDa endogenous cysteine proteinase inhibitor and an important biomarker of renal function. However, expressing recombinant cystatin C is difficult because of low yield and inclusion bodies in Escherichia coli (E. coli). In this study, we cloned HCC gene into...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2015.03.006
更新日期:2015-07-01 00:00:00
abstract::To obtain strong bacterial expression of proteins that seem to be hard to express in bacteria or are highly toxic for bacteria, it is possible to create a palette of similar constructs, differing only by several nucleotides, gradually deleted from the full-length clone by exonuclease III. When a construct is equipped ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2004.12.013
更新日期:2005-05-01 00:00:00
abstract::Granulins (GRNs) are potent growth factors that are upregulated in many aggressive cancers from a wide range of organs. GRNs form tight, disulphide bonded, beta hairpin stacks, making them difficult to express in recombinant form. We recently described Ov-GRN-1, a GRN family member secreted by the carcinogenic liver f...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2011.06.018
更新日期:2011-10-01 00:00:00
abstract::A high-expression plasmid, pKSGHE3-1, containing the salt-tolerant glutaminase (EC 3.5.1.2) from marine bacterium Micrococcus luteus K-3 was constructed. pKSGHE3-1 was made by inserting the DNA fragment (1.43 kb) containing the structural gene synthesized by polymerase chain reaction into the downstream region of the ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1998.1005
更新日期:1999-03-01 00:00:00
abstract::We present a new streptavidin-binding peptide for both the purification and the detection of recombinant proteins. The peptide possesses nanomolar-affinity for streptavidin and therefore was termed Nano-tag. The Nano-tag(15) is 15 amino acids long and binds to streptavidin with a dissociation constant of 4 nM and the ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2003.08.014
更新日期:2004-01-01 00:00:00
abstract::Lactase deficiency problem discourages many adults from consuming milk as a major source of micro- and macronutrients. Enzymatic hydrolysis of lactose is an ideal solution for this problem but such processing adds significant costs. In this study, a cold active β-galactosidase from Planococcus sp-L4 (bgal) was optimiz...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2015.09.008
更新日期:2016-09-01 00:00:00
abstract::The covalent addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) groups to lipid A, which resides in the outer membranes of bacteria such as Salmonella typhimurium and Escherichia coli, is the final step in the polymyxin-resistance pathway in these organisms. This modification is catalyzed by the inner membrane protein ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2005.08.028
更新日期:2006-03-01 00:00:00
abstract::A tremendous increase has taken place over the last decades in the biochemical and clinical use of antibodies. Unfortunately, the constantly growing demand has not been matched by a corresponding easy access to pure immunoglobulin, as purification procedures tend to be either laborious, expensive, or inefficient. We p...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/s1046-5928(03)00130-x
更新日期:2003-08-01 00:00:00
abstract::Transmembrane and coiled-coil domains 1 (TMCO1) has a highly conserved amino acid sequence among species, indicating a critical role of TMCO1 in cell physiology. The deficiency of TMCO1 in humans is associated with cerebrofaciothoracic dysplasia (CFTD), glaucoma, osteogenesis and the occurrence of cancer. TMCO1 was re...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2020.105803
更新日期:2021-03-01 00:00:00
abstract::A soluble N-terminal domain of the Escherichia coli adenylyl transferase (ATase) is responsible for deadenylylation activity of the intact enzyme. Previous studies of the deadenylylation activity have involved a fragment, AT-N423 (residues 1 to 423), which was extended by 17 amino acids to give AT-N440. This new domai...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2003.11.001
更新日期:2004-03-01 00:00:00
abstract::The fluorescent reporter enhanced Green Fluorescent Protein (EGFP) has been used for assaying a wide range of biological activities ranging from gene expression, or localization of target proteins through to intermolecular interactions. However, over-production of this protein in Escherichia coli has resulted in the p...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2011.05.003
更新日期:2011-10-01 00:00:00
abstract::The present report documents an improved enzyme assay method for the mammalian L-alanine:4,5-dioxovalerate transaminase which is of significant utility in work with crude tissue homogenates, cell cultures, or purified enzyme preparations. We also describe a new and rapid purification procedure for this enzyme from rat...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1994.1072
更新日期:1994-12-01 00:00:00
abstract::A putative laccase gene (lacG) from Geobacillus sp. JS12 was cloned and expressed as a fusion protein with six histidine residues in Escherichia coli BL21 (DE3) cells, and the protein was primarily found in inclusion bodies. The resulting insoluble proteins were solubilized with 6 M guanidine HCl and refolded using an...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2020.105646
更新日期:2020-09-01 00:00:00
abstract::Molecular-level investigation of proteins is increasingly important to researchers trying to understand the mechanisms of signal transmission. Heterotrimeric G proteins control the activation of many critical signal transmission cascades and are also implicated in numerous diseases. As part of a longer-term investigat...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2012.06.003
更新日期:2012-08-01 00:00:00
abstract::The role of FIC (Filamentation induced by cAMP)(2) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylation), in microorganisms, higher eukaryotes, and plants is emerging. The identity and function of FIC domain containin...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2012.08.020
更新日期:2012-11-01 00:00:00
abstract::Over-expression of heterologous proteins in Escherichia coli is commonly hindered by the formation of inclusion bodies. Nevertheless, refolding of proteins in vitro has become an essential requirement in the development of structural genomics (proteomics) and as a means of recovering functional proteins from inclusion...
journal_title:Protein expression and purification
pub_type: 杂志文章,评审
doi:10.1016/j.pep.2007.05.014
更新日期:2007-10-01 00:00:00
abstract::A long-lasting recombinant human albumin-linker-erythropoietin (EPO) is a human albumin gene fused to the N-terminal of EPO with a (GGSGG)(n)-repeated linker inserted between albumin and EPO. Albumin-EPO fusion genes were co-transfected with the dhfr gene. Albumin-EPO fusion protein has three kinds of sub-types (IALE,...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2009.07.003
更新日期:2009-12-01 00:00:00
abstract::Glycine N-acyltransferase (GLYAT) is a phase II metabolic detoxification enzyme for exogenous (xenobiotic) and endogenous carboxylic acids; consisting of fatty acids, benzoic acid, and salicylic acid. GLYAT catalyzes the formation of hippurate (N-benzoylglycine) from the corresponding glycine and benzoyl-CoA. Herein, ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2014.02.007
更新日期:2014-05-01 00:00:00
abstract::We examined expression of two plant genes encoding coclaurine N-methyltransferase (CMT) and norcoclaurine synthase (NCS) in Escherichia coli from the Salmonella entericaprpBCDE promoter (P(prpB)) and compared it to that from the strongest IPTG-inducible promoter, P(T7). In contrast to our previous study showing slight...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2008.06.008
更新日期:2008-10-01 00:00:00
abstract::The complement regulatory protein (CRP) of Trypanosoma cruzi is a developmentally regulated glycosylphosphatidylinositol (GPI)-anchored membrane protein that protects the parasite from complement-mediated killing, and is an important virulence determinant of the microorganism. CRP binds human complement components C3b...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/s1046-5928(02)00562-4
更新日期:2003-01-01 00:00:00
abstract::Bovine pancreatic procarboxypeptidase A has been overexpressed in a soluble and activatable form in Escherichia coli. When the protein was expressed under the control of bacteriophage T7 promoter in E. coli ADA494 (a thioredoxin reductase deficient bacteria), a thioredoxin fusion protein was produced at relatively hig...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/s1046-5928(02)00573-9
更新日期:2003-02-01 00:00:00
abstract::The sequence of the gene from Nocardia sp. NRRL 5646 encoding GTP cyclohydrolase I (GCH), gch, and its adjacent regions was determined. The open reading frame of Nocardia gch contains 684 nucleotides, and the deduced amino acid sequence represents a protein of 227 amino acid residues with a calculated molecular mass o...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2004.02.008
更新日期:2004-06-01 00:00:00
abstract::The cDNA for the alpha i1 protein that had undergone site-directed mutagenesis to change glycine-2 to alanine was ligated into a baculovirus transfer vector. A recombinant virus was obtained by transfecting Sf9 cells with both the wild-type baculovirus DNA and the transfer vector and screening for recombinant plaques....
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1993.1010
更新日期:1993-02-01 00:00:00
abstract::Cardiomyopathy-related mutations in human cardiac troponin subunits, including troponin C (hcTnC), troponin I (hcTnI), and troponin T (hcTnT), are well-documented. Recently, it has been recognised that human cardiac troponin (hcTn) is a sophisticated allosteric system. Therefore, the effect of drugs on this protein co...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2012.10.005
更新日期:2013-02-01 00:00:00