Process parameters optimization to produce the recombinant protein CFP10 for the diagnosis of tuberculosis.


:The aim of this study was to evaluate the parameters that affect the production of the recombinant 10 kDa culture filtrate protein (CFP10), a promising reagent of high specificity for intradermoreaction and other antigen-based methods used in the diagnosis of tuberculosis. Conditions of Escherichia coli growth temperature, induction temperature and IPTG-inducer concentration were evaluated in shake flasks and dissolved O2 concentrations of 15 and 30% were evaluated in a bioreactor. The process parameters defined on small scale were: growth temperature between 30 and 37 °C, induction temperature of 26 °C and IPTG concentration of 0.12 mM. The process conducted with 15% dissolved O2 presented a recombinant protein yield of 78.6 mg g-1 biomass and a proportion of recombinant protein (insoluble fraction) in relation to total insoluble protein of 72%, at the time of maximum productivity. The operation with 30% dissolved O2 resulted in lower recombinant protein yields of 62.9 mg g-1 biomass and 20% in relation to total insoluble protein, but in higher overall concentration in the culture broth (69.2 mg L-1versus 48.3 mg L-1). The protein identity was confirmed by mass spectrometry, showing high similarity to CFP10, 10 kDa of Mycobacterium tuberculosis H37Rv (score 95), and the purified antigen presented reactivity by the Western blotting assay.


Protein Expr Purif


Dela Coletta Troiano Araújo L,Wibrantz M,Rodríguez-Fernández DE,Karp SG,Talevi AC,Maltempi de Souza E,Soccol CR,Thomaz-Soccol V




Has Abstract


2019-02-01 00:00:00












  • Enhancing the soluble expression of an amylase in Escherichia coli by the mutations related to its domain interactions.

    abstract::The sequence and structure of the target protein exert a marked effect on its soluble expression in Escherichia coli. The effects of the mutation of an amylase isolated from Bacillus licheniformis (BLA) on its soluble expression in E. coli were investigated. A random mutation library of BLA was constructed to screen f...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Wang P,Qin W,Xu J,Yan Y,Tian J,Wu N,Yao B

    更新日期:2016-04-01 00:00:00

  • Isolation, overexpression, and biochemical characterization of the two isoforms of glutamic acid decarboxylase from Escherichia coli.

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    journal_title:Protein expression and purification

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    authors: De Biase D,Tramonti A,John RA,Bossa F

    更新日期:1996-12-01 00:00:00

  • Large-scale purification and characterization of recombinant fibroblast growth factor-saporin mitotoxin.

    abstract::In order to produce sufficient quantities of fibroblast growth factor-saporin (rFGF-2-SAP) mitotoxin for preclinical evaluation in models of diseases such as cancer and restenosis, we have undertaken the large-scale expression, purification, and characterization of the recombinant molecule. The fusion gene encoding rF...

    journal_title:Protein expression and purification

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    authors: McDonald JR,Ong M,Shen C,Parandoosh Z,Sosnowski B,Bussell S,Houston LL

    更新日期:1996-08-01 00:00:00

  • Refolding and characterization of a yeast dehydrodolichyl diphosphate synthase overexpressed in Escherichia coli.

    abstract::Dehydrodolichyl diphosphate synthase (DDPPs) catalyzes the sequential condensation of isopentenyl diphosphate with farnesyl diphosphate to synthesize long-chain dehydrodolichyl diphosphate, which serves as a precursor of glycosyl carrier in glycoprotein biosynthesis in eukaryotes. To perform kinetic and structural stu...

    journal_title:Protein expression and purification

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    authors: Chang SY,Tsai PC,Tseng CS,Liang PH

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  • Rapid purification of recombinant betaB2-crystallin using hydrophobic interaction chromatography.

    abstract::BetaB2-crystallin, the major subunit of beta-crystallins, is difficult to purify either from lens homogenate or from betaH-or betaL-crystallins. It has been prepared by heterologous expression in Escherichia coli. Most often, the methods used for purifying a recombinant globular protein employ the combination of ion-e...

    journal_title:Protein expression and purification

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    authors: Jobby MK,Sharma Y

    更新日期:2003-03-01 00:00:00

  • Plasma fibronectin: three steps to purification and stability.

    abstract::Large amounts of soluble fibronectin were easily purified from cryoprecipitated or fresh citrated human blood plasma by a three-step combination of gelatin and heparin-cellufine affinity chromatography. The elution conditions were optimized to obtain a homogeneous fraction on SDS-PAGE and Western blot under reducing c...

    journal_title:Protein expression and purification

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    authors: Poulouin L,Gallet O,Rouahi M,Imhoff JM

    更新日期:1999-10-01 00:00:00

  • High-level extracellular production of Rhizopus oryzae lipase in Pichia pastoris via a strategy combining optimization of gene-copy number with co-expression of ERAD-related proteins.

    abstract::Rhizopus oryzae lipase (ROL) is an important industrial enzyme limited in application due to its low production in native strains. Here, we used a new combined strategy to overexpress ROL in Pichia pastoris. An efficient method based on bio-brick was developed to construct a series of vectors harboring different copy ...

    journal_title:Protein expression and purification

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    authors: Jiao L,Zhou Q,Su Z,Xu L,Yan Y

    更新日期:2018-07-01 00:00:00

  • A four-step, inexpensive protocol for large-scale purification of goat uterine estrogen receptor.

    abstract::A relatively inexpensive yet highly efficient and extremely rapid procedure has been developed for the isolation and purification of estrogen receptor from the goat uterine cytosol. Greater than 1 mg of purified receptor protein could be obtained from 75 g of uterine tissue within a period of < 24 h, following this pr...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Zafar A,Thampan RV

    更新日期:1993-12-01 00:00:00

  • A single-step purification of biologically active recombinant human interleukin-5 from a baculovirus expression system.

    abstract::Recombinant human interleukin-5 (rhIL-5) was expressed in baculovirus-infected insect cells and purified to homogeneity from the culture medium in a single chromatographic step. Beginning with a cDNA encoding the full-length precursor form of human IL-5, including the authentic secretory leader sequence, recombinant b...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Brown PM,Scheid MP,O'Neill GP,Tagari PC,Nicholson DW

    更新日期:1995-02-01 00:00:00

  • Purification of major lignin peroxidase isoenzymes from Phanerochaete chrysosporium by chromatofocusing.

    abstract::The basidiomycete Phanerochaete chrysosporium produces several isoforms of lignin peroxidase, which catalyzes the oxidative depolymerization of lignin To date, ion-exchange chromatography and preparative isoelectric focusing (IEF) have been commonly used for isolation of lignin peroxidase isoenzymes. In this work we h...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Ollikka P,Leppänen VM,Anttila T,Suominen I

    更新日期:1995-06-01 00:00:00

  • Overexpression, oxidative refolding, and zinc binding of recombinant forms of the murine S100 protein MRP14 (S100A9).

    abstract::Recombinant murine MRP14 (mMRP14) was produced in Escherichia coli using the pGEX expression system. The mass of fusion protein, by electrospray ionization-mass spectrometry (ESI/MS), was 39,213 Da which compares well with the theoretical mass (39,210.4 Da). Thrombin digestion of fusion protein was expected at a clone...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Raftery MJ,Collinson L,Geczy CL

    更新日期:1999-03-01 00:00:00

  • Rapid purification method for human SFPQ by implementing zinc-induced polymerization.

    abstract::Splicing factor proline- and glutamine-rich (SFPQ) is an RNA-binding protein, playing significant roles in gene regulation and subnuclear body formation. Our recent serendipitous discovery showed that SFPQ binds zinc directly and forms an infinite polymer that is induced by zinc binding to the protein. The zinc-induce...

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    authors: Lim YW,Lee M

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  • Purification and characterization of human dehydrodolychil diphosphate synthase (DHDDS) overexpressed in E. coli.

    abstract::Protein asparagine (N)-linked glycosylation is a post-translational modification that occurs in the endoplasmic reticulum; it plays an important role in protein folding, oligomerization, quality control, sorting, and transport. Accordingly, disorders of glycosylation may affect practically every organ system. Dehydrod...

    journal_title:Protein expression and purification

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    authors: Giladi M,Edri I,Goldenberg M,Newman H,Strulovich R,Khananshvili D,Haitin Y,Loewenstein A

    更新日期:2017-04-01 00:00:00

  • Expression and purification of active recombinant human bikunin in Pichia pastoris.

    abstract::Bikunin is a proteoglycan exhibiting broad-spectrum inhibitory activity against serine proteases and could potentially suppress tumor cell invasion and metastasis. Here, we have successfully expressed recombinant human bikunin (rh-bikunin) in the methylotrophic yeast Pichia pastoris and established the purification pr...

    journal_title:Protein expression and purification

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    authors: Jian-qiu W,Feng-qin Y,Dou-dou W,Lei B,Nan S,Cong-yan L,Ting Z,Wei-qun Y

    更新日期:2008-08-01 00:00:00

  • Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions.

    abstract::Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusi...

    journal_title:Protein expression and purification

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    authors: Liu L,Spurrier J,Butt TR,Strickler JE

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  • Purification and structural characterization of porcine L-threonine dehydrogenase.

    abstract::L-Threonine dehydrogenase was purified 10,000-fold to a specific activity approximately 300 protein from porcine liver mitochondria. Purification to apparent homogeneity was achieved by sequential chromatography on DEAE Sepharose FF, Affi-Gel Blue, Sephacryl S-200, Matrex Gel Red A, and Matrex Gel Gre...

    journal_title:Protein expression and purification

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    authors: Kao YC,Davis L

    更新日期:1994-10-01 00:00:00

  • Expression of the extracellular domain of the human heat-stable enterotoxin receptor in Escherichia coli and generation of neutralizing antibodies.

    abstract::The entire extracellular domain of the human heat-stable enterotoxin (ST) receptor as well as a truncated N-terminal domain were cloned as glutathione S-transferase fusion proteins and expressed in Escherichia coli. The recombinant fusion proteins were purified from both the cytosol and the inclusion body fractions by...

    journal_title:Protein expression and purification

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    authors: Nandi A,Mathew R,Visweswariah SS

    更新日期:1996-09-01 00:00:00

  • CRISPR-Cas9 mediated genetic engineering for the purification of the endogenous integrator complex from mammalian cells.

    abstract::The Integrator Complex (INT) is a large multi-subunit protein complex, containing at least 14 subunits and a host of associated factors. These protein components have been established through pulldowns of overexpressed epitope tagged subunits or by using antibodies raised against specific subunits. Here, we utilize CR...

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    authors: Baillat D,Russell WK,Wagner EJ

    更新日期:2016-12-01 00:00:00

  • Purification and biochemical characterization of the ErmSF macrolide-lincosamide-streptogramin B resistance factor protein expressed as a hexahistidine-tagged protein in Escherichia coli.

    abstract::The erm proteins confer resistance to the MLS (macrolide-lincosamide-streptogramin B) antibiotics in various microorganisms, including pathogens, through dimethylation of a single adenine residue (A2085: Bacillus subtilis coordinate) of the 23S rRNA to reduce the affinity of antibiotics, thereby enabling the cells to ...

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    authors: Jin HJ,Yang YD

    更新日期:2002-06-01 00:00:00

  • Identification, expression, and purification of a unique stable domain from human HSPC144 protein.

    abstract::HSPC144 is a newly identified gene in human CD34(+) hematopoietic stem/progenitor cells. In this work, we have expressed and purified the 225-residue protein from Escherichia coli BL21 (DE3) and identified a stable fragment HSPC144-P (residues 44-225) by limited proteolysis method. The HSPC144-P fragment exhibits high...

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    authors: Song AX,Chang YG,Gao YG,Lin XJ,Shi YH,Lin DH,Hang QH,Hu HY

    更新日期:2005-07-01 00:00:00

  • Expression and structural characterization of human translocase of inner membrane of mitochondria Tim50.

    abstract::The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim50 is a major receptor in TIM23 complex, which spans the inner membrane with a single transmembrane segment and exposes a large hydrophilic domain in the interme...

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    authors: Zhang Y,Xu Y,Zhao Q,Ji Z,Li Q,Li SJ

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  • Characterization of recombinant pectate lyase refolded from inclusion bodies generated in E. coli BL21(DE3).

    abstract::Pectate lyase (EC gene from Bacillus subtilis RCK was cloned and expressed in Escherichia coli to maximize its production. In addition to soluble fraction, bioactive pectate lyase was also obtained from inclusion body aggregates by urea solubilization and refolding under in vitro conditions. Enzyme with speci...

    journal_title:Protein expression and purification

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    authors: Kumar S,Jain KK,Singh A,Panda AK,Kuhad RC

    更新日期:2015-06-01 00:00:00

  • Improved soluble expression of a single-chain antibody fragment in E. coli for targeting CA125 in epithelial ovarian cancer.

    abstract::Production of antibody fragments in heterologous hosts such as Escherichiacoli provides a unique and cost-effective method to develop engineered vectors for tumor targeting. A single-chain Fragment variable (scFv) of the murine monoclonal antibody MAb-B43.13 targeting CA125 in epithelial ovarian cancer was previously ...

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    authors: Sharma SK,Suresh MR,Wuest FR

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  • Design and preparation of non-tagged Yersinia pestis LcrV antigen in Escherichia coli and its immunogenicity in BALB/c mice.

    abstract::The whole encoding sequence for Yersinia pestis LcrV antigen was cloned into pET-32a(+) and expressed in Escherichia coli BL21 (DE3). The LcrV was high level expressed in the E. coli cytoplasm in a completely soluble form. Recombinant LcrV could be purified from the supernatant of the bacteria lysate after chromatogra...

    journal_title:Protein expression and purification

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    authors: li J,Li B,Li G,Ren J,Zhang J,Xu C,Yang X,Liu S,Fu L,Chen W

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  • Generation and evaluation of mammalian secreted and membrane protein expression libraries for high-throughput target discovery.

    abstract::Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications....

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    authors: Panavas T,Lu J,Liu X,Winkis AM,Powers G,Naso MF,Amegadzie B

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  • Cloning, expression, purification, and biochemical characterisation of the FIC motif containing protein of Mycobacterium tuberculosis.

    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...

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    authors: Mishra S,Bhagavat R,Chandra N,Vijayarangan N,Rajeswari H,Ajitkumar P

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  • Co-expression of human protein disulfide isomerase (hPDI) enhances secretion of bovine follicle-stimulating hormone (bFSH) in Pichia pastoris.

    abstract::Bovine follicle-stimulating hormone (bFSH) is a pituitary gonadotropin composed of two non-covalently associated polypeptide subunits, which must be glycosylated, folded, and assembled as a heterodimer to be biologically active. Low-level expression of the recombinant bFSH is the factor that limits its usefulness as a...

    journal_title:Protein expression and purification

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    authors: Huo X,Liu Y,Wang X,Ouyang P,Niu Z,Shi Y,Qiu B

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  • Expression, purification, crystallization, and preliminary X-ray analysis of the N-terminal domain of Escherichia coli adenylyl transferase.

    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


    authors: Xu Y,Wen D,Clancy P,Carr PD,Ollis DL,Vasudevan SG

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    abstract::The high specific lysyl endopeptidase (Lys-C; EC is often used for the initial fragmentation of polypeptide chains during protein sequence analysis. However, due to its specificity it could be a useful tool for the production of tailor-made protein hydrolysates with for example bioactive or techno functiona...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Stressler T,Eisele T,Meyer S,Wangler J,Hug T,Lutz-Wahl S,Fischer L

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    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


    authors: Zhang N,Tang M,Wen M,Cao Y,OuYang B

    更新日期:2021-03-01 00:00:00