当前位置： SCI文献检索 > PROTEIN EXPRESSION AND PURIFICATION期刊下所有文献 > Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells.

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells.

Abstract：

:Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody-antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1-20mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS-PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Zhao Y,Gutshall L,Jiang H,Baker A,Beil E,Obmolova G,Carton J,Taudte S,Amegadzie B

doi

10.1016/j.pep.2009.04.012

subject

Has Abstract

pub_date

2009-10-01 00:00:00

pages

182-9

issue

2

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(09)00102-8

journal_volume

67

pub_type

杂志文章

相关文献

PROTEIN EXPRESSION AND PURIFICATION文献大全
  • A novel method for increasing the expression level of recombinant proteins.

    abstract：:Expression of recombinant proteins is an important step towards elucidating the functions of many genes discovered through genomic sequencing projects. It is also critical for validating gene targets and for developing effective therapies for many diseases. Here we describe a novel method to express recombinant protei...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/s1046-5928(03)00075-5

    authors： Wang A,Clapper J,Guderian JA,Foy TM,Fanger GR,Retter MW,Skeiky YA

    更新日期：2003-07-01 00:00:00

  • Codon engineering for improved antibody expression in mammalian cells.

    abstract：:While well established in bacterial hosts, the effect of coding sequence variation on protein expression in mammalian systems is poorly characterized outside of viral proteins or proteins from distant phylogenetic families. The potential impact is substantial given the extensive use of mammalian expression systems in ...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2007.05.017

    authors： Carton JM,Sauerwald T,Hawley-Nelson P,Morse B,Peffer N,Beck H,Lu J,Cotty A,Amegadzie B,Sweet R

    更新日期：2007-10-01 00:00:00

  • Overproduction, in Escherichia coli, of soluble taxadiene synthase, a key enzyme in the Taxol biosynthetic pathway.

    abstract：:Taxadiene synthase catalyzes the conversion of the universal precursor of diterpenoids, geranylgeranyl diphosphate, to taxadiene, a key intermediate in Taxol (paclitaxel) biosynthesis. The gene encoding taxadiene synthase was cloned recently. Here we report a method for the heterologous overexpression of cDNA encoding...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1006/prep.1998.0870

    authors： Huang KX,Huang QL,Wildung MR,Croteau R,Scott AI

    更新日期：1998-06-01 00:00:00

  • Heterologous production, purification and characterization of enzymatically active Sindbis virus nonstructural protein nsP1.

    abstract：:Alphavirus nonstructural protein nsP1 possesses distinct methyltransferase (MTase) and guanylyltransferase (GTase) activities involved in the capping of viral RNAs. In alphaviruses, the methylation of GTP occurs before RNA transguanylation and nsP1 forms a covalent complex with m(7)GMP unlike the host mRNA guanylyltra...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2011.05.022

    authors： Tomar S,Narwal M,Harms E,Smith JL,Kuhn RJ

    更新日期：2011-10-01 00:00:00

  • Tabtoxin-resistant protein: overexpression, purification, and characterization.

    abstract：:One of the self-protection mechanisms in Pseudomonas syringae pv. tabaci, a pathogen of tobacco wildfire, is thought to be due to its tabtoxin-resistance gene (ttr). In this study, the ttr gene was inserted into an expression vector, pQE30, and successfully expressed in Escherichia coli M15 at high levels. The purifie...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1006/prep.2001.1586

    authors： Liu J,Le Y,Ye B,Zhen Y,Zhu C,Shen J,Zhang R

    更新日期：2002-04-01 00:00:00

  • Expression in E. coli and purification of the nucleoside diphosphate kinase b from Leishmania major.

    abstract：:Leishmaniasis is considered by the World Health Organization to be the second most important disease caused by a protozoan parasite. Biochemical and molecular biology studies can help in the understanding of the biology of the Leishmania parasite. All protozoan parasites, including Leishmania, are unable to synthesize...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2006.05.010

    authors： de Oliveira AH,Ruiz JC,Cruz AK,Greene LJ,Rosa JC,Ward RJ

    更新日期：2006-10-01 00:00:00

  • Optimization of human D-amino acid oxidase expression in Escherichia coli.

    abstract：:Human D-amino acid oxidase (hDAAO) is a flavoprotein that plays a key role in the pathophysiology of schizophrenia. So far, the biochemical characterization of this enzyme has been hampered by the difficulty of expressing it in a common heterologous host such as Escherichia coli. Increasing amounts of recombinant hDAA...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2009.05.013

    authors： Romano D,Molla G,Pollegioni L,Marinelli F

    更新日期：2009-11-01 00:00:00

  • Cloning, expression, purification, and characterization of Nocardia sp. GTP cyclohydrolase I.

    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

    authors： He A,Simpson DR,Daniels L,Rosazza JP

    更新日期：2004-06-01 00:00:00

  • Expression of mammalian Rab Escort protein-1 and -2 in yeast Saccharomyces cerevisiae.

    abstract：:Rab GTPases are post-translationally geranylgeranylated on their C-terminal cysteines by Rab geranylgeranyl transferase (RabGGTase) and this modification is essential for their biological activity. Rab Escort Protein (REP) is a molecular chaperone that assists in the prenylation reaction carried out by RabGGTase. Muta...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/s1046-5928(02)00506-5

    authors： Sidorovitch V,Niculae A,Kan N,Ceacareanu AC,Alexandrov K

    更新日期：2002-10-01 00:00:00

  • Expression and purification of receptor for activated C-kinase 1 (RACK1).

    abstract：:Receptor for activated C-kinase (RACK1) binds to protein kinase C and functions as an anchor for several other cellular components. Most in vitro studies of RACK1 have been carried out with RACK1 fused to a soluble fusion protein partner, such as GST or MBP. Here, we show that fusion complexes may exist as large solub...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/s1046-5928(03)00135-9

    authors： Bjørndal B,Trave G,Hageberg I,Lillehaug JR,Raae AJ

    更新日期：2003-09-01 00:00:00

  • Expression and purification of recombinant M-Pol I from Saccharomyces cerevisiae with alpha-1,6 mannosylpolymerase activity.

    abstract：:Mannan outer chain N-glycan structures are yeast/fungal-specific typically found on secreted and cell wall glycoproteins. Mannan outer chains consist of an alpha-1,6 polymannose backbone attached to a Man(8-10)(GlcNAc)(2) core. The backbone contains branches of alpha-1,2 mannose residues, terminated with alpha-1,3 man...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2009.02.013

    authors： Rodionov D,Romero PA,Berghuis AM,Herscovics A

    更新日期：2009-07-01 00:00:00

  • Comparative analysis of native and cysteine-deficient HIV-1 reverse transcriptase.

    abstract：:To study the subunit structure and the active site of human immunodeficiency virus reverse transcriptase (RT), the enzyme was expressed in E. coli and purified to homogeneity in large quantities. The recombinant enzyme consists of two major polypeptides of 66,000 and 53,000 Da in equimolar amounts and a minor species ...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/1046-5928(92)90005-h

    authors： Fischer M,Lifshitz R,Katz T,Liefer I,Ben-Artzi H,Gorecki M,Panet A,Zeelon E

    更新日期：1992-08-01 00:00:00

  • Purification of recombinant chimeric B72.3 Fab' and F(ab')2 using streptococcal protein G.

    abstract：:Streptococcal protein G has been used extensively for the purification of antibodies using the interaction of the Fc region with protein G. Many antibodies also interact with protein G through a low-affinity binding site for the Fab region. The exploitation of this low-affinity interaction for the purification of Fab'...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/s1046-5928(05)80037-3

    authors： Proudfoot KA,Torrance C,Lawson AD,King DJ

    更新日期：1992-10-01 00:00:00

  • Improvements to the throughput of recombinant protein expression in the baculovirus/insect cell system.

    abstract：:Recombinant baculoviruses have proved to be a very useful means to express many proteins over the last 20 years. Since their introduction, there have been a number of significant improvements that have simplified and speeded up the construction of baculoviruses. One of the most commonly used methods relies upon recomb...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2005.03.021

    authors： McCall EJ,Danielsson A,Hardern IM,Dartsch C,Hicks R,Wahlberg JM,Abbott WM

    更新日期：2005-07-01 00:00:00

  • Development of stable isotope and selenomethionine labeling methods for proteins expressed in Pseudomonas fluorescens.

    abstract：:Pseudomonas fluorescens is a robust protein expression system that is very well suited for high throughput protein expression for structural genomics studies. Since NMR spectroscopy and X-ray crystallography are both used by various investigators in structure elucidation studies, the availability of target proteins la...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2008.12.012

    authors： Madduri K,Badger M,Li ZS,Xu X,Thornburgh S,Evans S,Dhadialla TS

    更新日期：2009-05-01 00:00:00

  • Production of recombinant Conkunitzin-S1 in Escherichia coli.

    abstract：:Conkunitzin-S1 from the cone snail Conus striatus is the first member of a new neurotoxin family with a canonical Kunitz domain fold. Conk-S1 is 60 amino acids long and lacks one of the three conserved disulfide bonds typically found in Kunitz domain modules. It binds specifically to voltage activated potassium channe...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2006.01.019

    authors： Bayrhuber M,Graf R,Ferber M,Zweckstetter M,Imperial J,Garrett JE,Olivera BM,Terlau H,Becker S

    更新日期：2006-06-01 00:00:00

  • The effects of L-arginine on protein stability and DNA binding ability of SaeR, a transcription factor in Staphylococcus aureus.

    abstract：:The SaeRS two-component system in Staphylococcus aureus controls the expression of a series of virulence factors, such as hemolysins, proteases, and coagulase. The response regulator, SaeR, belongs to the OmpR family with an N-terminal regulatory domain and a C-terminal DNA binding domain. To improve the production an...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2020.105765

    authors： Fan R,Shi X,Guo B,Zhao J,Liu J,Quan C,Dong Y,Fan S

    更新日期：2021-01-01 00:00:00

  • Overexpression and purification of the Escherichia coli inner membrane enzyme acyl-acyl carrier protein synthase in an active form.

    abstract：:Acyl-acyl carrier protein synthase (Aas) is widely used to synthesize thioester adducts of fatty acids between 8 and 18 carbons in length enzymatically to the phosphopantetheine group of acyl carrier protein. The enzyme is an 80.6-kDa inner membrane protein that functions in vivo as a 2-acylglycerophosphoethanolamine ...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1006/prep.2000.1206

    authors： Shanklin J

    更新日期：2000-04-01 00:00:00

  • Heterologous expression and pro-peptide supported refolding of the high specific endopeptidase Lys-C.

    abstract：:The high specific lysyl endopeptidase (Lys-C; EC 3.4.21.50) 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

    doi：10.1016/j.pep.2015.09.024

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

    更新日期：2016-02-01 00:00:00

  • An improved method for high-level soluble expression and purification of recombinant amyloid-beta peptide for in vitro studies.

    abstract：:Amyloid-beta (Aβ) peptide mediates several neurodegenerative diseases. The 42 amino acid (Aβ1-42) is the predominant form of peptide found in the neuritic plaques and has been demonstrated to be neurotoxic in vivo and in vitro. The availability of large quantities of Aβ peptide will help in several biochemical and bio...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2015.05.015

    authors： Chhetri G,Pandey T,Chinta R,Kumar A,Tripathi T

    更新日期：2015-10-01 00:00:00

  • Expression, purification, and functional analysis of the TyrR protein of Haemophilus influenzae.

    abstract：:The gene that was inferred to encode the TyrR protein of Haemophilus influenzae Rd was synthesized by polymerase chain reaction and inserted into a T7-based expression vector. Methods were developed to overexpress the TyrR protein of H. influenzae in Escherichia coli and to purify the protein on a large scale. Both in...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1006/prep.1997.0757

    authors： Zhu Q,Zhao S,Somerville RL

    更新日期：1997-07-01 00:00:00

  • Solubility of the catalytic domains of Botulinum neurotoxin serotype E subtypes.

    abstract：:The Clostridium botulinum neurotoxins (BoNTs) are the most potent protein toxins known to humans. There are seven serotypes of the BoNTs (A-G), among which serotypes A, B, E and F are known to cause natural human intoxication. To date, eleven subtypes of LC/E, termed E1∼E11, have been identified. The LCs of BoNT/E wer...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2015.10.003

    authors： Chen S,Barbieri JT

    更新日期：2016-02-01 00:00:00

  • Improvements of a high-throughput protein purification process using a calcium-dependent setup.

    abstract：:The Human Secretome Project aims to produce and purify all human secreted proteins as full-length. In order to enable this, a robust, gentle and effective purification process is needed, where multiple proteins can be purified in parallel. For this reason, a purification system based on a Protein C-tag and the HPC4 an...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2020.105698

    authors： Kanje S,Enstedt H,Dannemeyer M,Uhlén M,Hober S,Tegel H

    更新日期：2020-11-01 00:00:00

  • Enhanced soluble expression of recombinant Flavobacterium heparinum heparinase I in Escherichia coli by fusing it with various soluble partners.

    abstract：:Heparinase I (HepA) was originally isolated from Flavobacterium heparinum (F. heparinum) and specifically cleaves heparin/heparan sulfate in a site-dependent manner, showing great promise for producing low molecular weight heparin (LMWH). However, expressing recombinant HepA is extremely difficult in Escherichia coli ...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2012.03.016

    authors： Huang J,Cao L,Guo W,Yuan R,Jia Z,Huang K

    更新日期：2012-06-01 00:00:00

  • Temperature, media, and point of induction affect the N-terminal processing of interleukin-1beta.

    abstract：:The expression of recombinant proteins in bacterial hosts may alter the biophysical properties of the protein of interest as a result of differences in post-translational processing from that observed when produced in the native cell. For example, recombinant human interleukin-1beta (IL-1beta) is produced as three iso...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2005.01.010

    authors： Covalt JC Jr,Cao TB,Magdaroag JR,Gross LA,Jennings PA

    更新日期：2005-05-01 00:00:00

  • Isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity.

    abstract：:The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Gl...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/s1046-5928(03)00015-9

    authors： Neyestani TR,Djalali M,Pezeshki M

    更新日期：2003-06-01 00:00:00

  • Homologous expression of the feruloyl esterase B gene from Aspergillus niger and characterization of the recombinant enzyme.

    abstract：:The faeB gene encoding the feruloyl esterase B (FAEB) was isolated from Aspergillus niger BRFM131 genomic DNA. The faeB gene, with additional sequence coding for a C-terminal histidine tag, was inserted into an expression vector under the control of the gpd promoter and trpC terminator and expressed in a protease defi...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2004.05.019

    authors： Levasseur A,Benoit I,Asther M,Asther M,Record E

    更新日期：2004-09-01 00:00:00

  • Peptidyl-prolyl isomerase and the biological activities of recombinant protein cyclophilin from Pyropia yezoensis (PyCyp).

    abstract：:Cyclophilins are highly conserved proteins associated with peptidyl-prolyl cis-trans isomerase activity (PPIase). The present study was designed to analyze the biological activity of recombinant cyclophilin from the marine red algae Pyropia yezoensis (PyCyp). The cyclophilin gene from P. yezoensis was cloned into the ...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2020.105636

    authors： Ulagesan S,Choi JW,Nam TJ,Choi YH

    更新日期：2020-08-01 00:00:00

  • Expression and purification of stable 33-kDa soluble human CD23 using the Drosophila S2 expression system.

    abstract：:CD23, a 45-kDa type II membrane glycoprotein present on B cells, monocytes, and other human immune cells, is a low-affinity receptor for IgE. The extracellular region of the membrane-bound human CD23 is processed into at least four soluble (s) CD23 forms, with apparent molecular masses of 37, 33, 29, and 25 kDa. High ...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1006/prep.2001.1448

    authors： Khandekar SS,Mayer RJ,Cusimano DM,Katchur SR,Appelbaum ER

    更新日期：2001-07-01 00:00:00

  • Improved performance of column chromatography by arginine: dye-affinity chromatography.

    abstract：:Arginine has been effectively used in various column chromatographies for improving recovery and resolution, and suppressing aggregation. Here, we have tested the effectiveness of arginine as an eluent in dye-affinity column chromatography using Blue-Sepharose, which binds enzymes requiring adenyl-containing cofactors...

    journal_title：Protein expression and purification

    pub_type： 杂志文章

    doi：10.1016/j.pep.2006.10.005

    authors： Arakawa T,Ejima D,Tsumoto K,Ishibashi M,Tokunaga M

    更新日期：2007-04-01 00:00:00