Abstract:
:Human lysosomal beta-hexosaminidase exists in two major forms: the A isoform is composed of both alpha and beta chains, while the B form is a homopolymer of beta chains. Deficiency of beta-hexosaminidase underlies the GM2 gangliosidoses. We have produced active beta-hexosaminidase B in cultured insect (Sf9) cells by isolation of a recombinant insect virus (baculovirus) containing the cDNA for the beta chain within the viral polyhedron gene and infection of Sf9 cells with this construct. That portion of the enzyme secreted into the medium, 50%, was purified with concanavalin A Sepharose and subsequent affinity chromatography to yield beta-hexosaminidase B that is 75% pure. The product has an N-terminal amino acid sequence, specific activity, and size (M(r) 62,000) similar to that of the enzyme present in cultured human fibroblasts. However, endo H sensitivity studies revealed that the oligosaccharide structures present on recombinant beta-hexosaminidase B differ from those found on the enzyme synthesized in the human system. In addition, these structures lack the mannose 6-phosphate recognition marker that targets degradative hydrolases to lysosomes. Despite these differences, recombinant beta-hexosaminidase B does serve as a specific substrate for the mannose phosphorylating enzyme, N-acetylglucosaminyl phosphotransferase. Furthermore, the oligosaccharide moieties phosphorylated in vitro match those phosphorylated in vivo, pointing to the conformational integrity of the recombinant enzyme. Generous amounts of easily obtained, easily purified, and properly folded beta-hexosaminidase B will facilitate physical structural analysis of the enzyme.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Boose JA,Tifft CJ,Proia RL,Myerowitz Rdoi
10.1016/1046-5928(90)90003-hsubject
Has Abstractpub_date
1990-11-01 00:00:00pages
111-20issue
2eissn
1046-5928issn
1096-0279journal_volume
1pub_type
杂志文章abstract::Soluble oligomers and fibrillar deposits of amyloid beta (Abeta) are key agents of Alzheimer's disease pathogenesis. However, the mechanism of amyloid aggregation and its interaction with live cells still remain unclear requiring the preparation of large amounts of pure and different Abeta peptides. Here we describe a...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2009.02.009
更新日期:2009-07-01 00:00:00
abstract::A family of restriction enzyme- and ligation-independent cloning vectors has been developed for producing recombinant His-tagged fusion proteins in Escherichia coli. These are based on pURI2 and pURI3 expression vectors which have been previously used for the successful production of recombinant proteins at the millig...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2010.10.013
更新日期:2011-03-01 00:00:00
abstract::Streptavidin is a versatile molecule for many in vitro and in vivo applications. To optimize the production of the full-length streptavidin in a soluble and functional form via secretion using Bacillus subtilis as the expression host, three different strategies were used. These strategies include the construction of a...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2001.1582
更新日期:2002-04-01 00:00:00
abstract::Staphylococcal enterotoxins (SEs) are powerful superantigens that stimulate non-specific T-cell proliferation produced by Staphylococcus aureus and draw considerable attention as ideal drugs for cancer therapy. The filtrate of S. aureus culture has been used as ampul named Staphylococcal enterotoxin C injection in cli...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.08.005
更新日期:2007-12-01 00:00:00
abstract::Enzymatic hydrolysis of the N-iminylamide was investigated in this study. An enzyme possessing N-iminylamidase activity from pig liver was purified to electrophoretic homogeneity. This enzyme was also active, however, with imides and appears to be identical to pig liver imidase. The identification was confirmed by cop...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2004.12.008
更新日期:2005-03-01 00:00:00
abstract::A full-length cDNA sequence of plant type CRY (designated Hae-P-CRY) was cloned from the green alga Haematococcus pluvialis. The cDNA sequence was 3608 base pairs (bp) in length, which contained a 2988-bp open reading frame encoding 995 amino acids with molecular mass of 107.7 kDa and isoelectric point of 6.19. Multip...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2020.105633
更新日期:2020-08-01 00:00:00
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 an...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2009.04.012
更新日期:2009-10-01 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::Structural biology places a high demand on proteins both in terms of quality and quantity. Although many protein expression and purification systems have been developed, an efficient and simple system which can be easily adapted is desirable. Here, we report a new system which combines improved expression, solubility ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2008.09.008
更新日期:2009-02-01 00:00:00
abstract::The entire stx2 region from Escherichia coli O157:H7, containing two open reading frames (stx2a and stx2b), was cloned into pET-32a with a single promoter, and transformed into E. coli BL21 (DE3) pLysS. We used two methods of IPTG induction using LB medium and auto-induction using ZYM-5052 medium to express recombinan...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2009.05.005
更新日期:2009-10-01 00:00:00
abstract::Wild-type and deglycosylated forms of human prostate-specific antigen were expressed in Chinese hamster ovary (CHO) cells as zymogens. ProPSA was collected from conditioned medium and purified using a single cation-exchange chromatographic step for the deglycosylated form and cation-exchange followed by gel filtration...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2000.1342
更新日期:2000-12-01 00:00:00
abstract::Ricin A chain (RTA) mutants which had been modified by the addition of three lysine residues, three lysines and an alanine, or six histidine residues at the carboxyl terminus were expressed in Escherichia coli. The recombinant proteins were purified to homogeneity by ion-exchange chromatography on CM-Sepharose CL-6B. ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1995.1087
更新日期:1995-10-01 00:00:00
abstract::l-glutamate oxidase (GLOD), encoded by the gox gene, catalyses the transformation of l-glutamic acid into α-ketoglutaric acid (α-KG). In the present study, Pichia pastoris was used for heterologous production of GLOD following optimization of the gox coding sequence for expression in the yeast host. A series of constr...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2016.09.014
更新日期:2017-01-01 00:00:00
abstract::Formation of biominerals often involves specific proteins that modulate the process of matrix assembly, nucleation, and crystal growth. AP7 is an aragonite-associated protein of 7 kDa and is intrinsically disordered. The structural disorder of AP7 makes it very difficult to express in Escherchiacoli. In this work, we ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2014.05.002
更新日期:2014-08-01 00:00:00
abstract::The purpose of this work was to characterize the functions of two sesquiterpene synthase genes from moso bamboo (Phyllostachys edulis). Two novel sesquiterpene synthase genes, belonging to the Tpsa subfamily, were isolated from moso bamboo. MoTPS2 was 1641 bp in length and encoded a protein of 63 kDa, whereas MoTPS6 w...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2015.11.019
更新日期:2016-04-01 00:00:00
abstract::An internal fragment (978 bp) corresponding to the dog zona pellucida glycoprotein-3 (DZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was amplified by polymerase chain reaction from a full-length cDNA clone. The amplified SacI and PstI restricted fragment was cloned in-fra...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1997.0869
更新日期:1998-04-01 00:00:00
abstract::NAD(+)-dependent malic enzyme (NAD-ME) gene from Escherichia coli K12 was inserted into an expression vector pET24b(+) and transformed into E. coli BL21 (DE3). Recombinant NAD-ME was expressed upon IPTG induction, purified with affinity chromatography, and biochemically characterized. The results showed that recombina...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2006.11.017
更新日期:2007-05-01 00:00:00
abstract::Hydrophobic charge-induction chromatography (HCIC) using 4-mercaptoethylpyridine (4-MEP) as the ligand is used to purify antibodies. The 4-MEP resin ligand has high affinity for antibodies, which makes it difficult to optimize the elution conditions. Recent studies showed that arginine is effective at eluting and puri...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2016.09.004
更新日期:2017-01-01 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::Mutant analogues of recombinant human immune interferon (IFN-gamma) with higher stability and biological activity were prepared. Depending on the analogue, protein structure modification might involve introduction of an intramonomer disulfide bond (through replacements of Glu7Cys and Ser69Cys), C-terminal shortening b...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2001.1565
更新日期:2002-03-01 00:00:00
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
doi:10.1006/prep.1998.1015
更新日期:1999-03-01 00:00:00
abstract::Plants produce a large number of cellulases that are either secreted or anchored in the plasma membrane where they likely function in various aspects of cellulose synthesis, modification and degradation during plant growth and development. Very few of these enzymes have been characterized in any detail, however. Here ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2012.08.009
更新日期:2012-10-01 00:00:00
abstract::The Ca2+-ATPase accounts for the majority of Ca2+ removed from the cytoplasm during cardiac muscle relaxation. The Ca2+-ATPase is regulated by phospholamban, a 52 amino acid phosphoprotein, which inhibits Ca2+-ATPase activity by decreasing the apparent affinity of the ATPase for Ca2+. To study the physical mechanism o...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2003.11.005
更新日期:2004-03-01 00:00:00
abstract::Developmentally regulated G-proteins (DRGs) are a highly conserved family of GTP-binding proteins found in archaea, plants, fungi and animals, indicating important roles in fundamental pathways. Their function is poorly understood, but they have been implicated in cell division, proliferation, and growth, as well as s...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2009.05.009
更新日期:2009-10-01 00:00:00
abstract::Strategies for the expression of precursors of eukaryotic secreted proteins as part of fused proteins in Escherichia coli have been explored. A fusion protein with beta-galactosidase at the N-terminal end and honeybee prepromelittin at the C-terminal end (beta-gal-pM) was expressed in low amounts as a cleaved polypept...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/1046-5928(91)90095-z
更新日期:1991-10-01 00:00:00
abstract::Using a codon-optimized gene fragment, we report remarkable yields for extracellular domain of human NK cell receptor (NKp30ex) when produced on M9 minimal medium, even with low (2g/L) glucose concentration. The yields were identical using media containing (15)NH(4)Cl or (15)NH(4)Cl in combination with all-(13)C-d-glu...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2012.09.016
更新日期:2012-12-01 00:00:00
abstract::An expression library was generated from a partial NcoI and HindIII digest of genomic DNA from the thermophilic bacterium, Bacillus stearothermophilus P1. The DNA fragments were cloned into the expression vector pQE-60 and transformed into Escherichia coli M15[EP4]. Sequence analysis of a lipase gene showed an open re...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2001.1456
更新日期:2001-08-01 00:00:00
abstract::Production of VCSM13 phage displaying a high density of CD147 ectodomain (CD147Ex) was achieved when culturing conditions were modulated. A phagemid expressing CD147Ex was constructed and used to produce phage display CD147Ex gpVIII fusion protein in TG1 Escherichia coli. Displaying of CD147Ex via gpVIII was successfu...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2003.08.019
更新日期:2003-12-01 00:00:00
abstract::The Ig-binding properties of protein L from Peptostreptococcus magnus and protein G from Streptococcus have been successfully combined through the construction of a novel hybrid protein, consisting of a single Ig-binding domain from each protein. The biophysical and biochemical properties of this construct have been c...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.11.007
更新日期:2008-03-01 00:00:00
abstract::Hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) was utilized for the separation of native human antithrombin (AT) and a partially denaturated form of AT, known as the latent form (L-AT). The AT used in this study is commercially available (Atenativ, Pharmacia & Upjohn, Sweden) and contains al...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2000.1349
更新日期:2001-02-01 00:00:00