Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

Abstract The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

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Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

Blood ◽

10.1182/blood.v84.4.1157.bloodjournal8441157 ◽

1994 ◽

Vol 84 (4) ◽

pp. 1157-1163 ◽

Cited By ~ 1

Author(s):

EA Barron-Casella ◽

TS Kickler ◽

OC Rogers ◽

JF Casella

Keyword(s):

Amino Acids ◽

Fusion Protein ◽

Fusion Proteins ◽

Disulfide Bonds ◽

Affinity Purification ◽

Platelet Glycoprotein ◽

Enzyme Linked Immunosorbent Assay ◽

Glutathione S Transferase ◽

Amino Terminal ◽

Posttransfusion Purpura

The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

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Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix.

The Journal of Cell Biology ◽

10.1083/jcb.123.1.119 ◽

1993 ◽

Vol 123 (1) ◽

pp. 119-126 ◽

Cited By ~ 111

Author(s):

W Voos ◽

B D Gambill ◽

B Guiard ◽

N Pfanner ◽

E A Craig

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Fusion Protein ◽

Fusion Proteins ◽

Dual Role ◽

Intermembrane Space ◽

Heme Binding ◽

Membrane Translocation ◽

Amino Terminal ◽

The Matrix

To test the hypothesis that 70-kD mitochondrial heat shock protein (mt-hsp70) has a dual role in membrane translocation of preproteins we screened preproteins in an attempt to find examples which required either only the unfoldase or only the translocase function of mt-hsp70. We found that a series of fusion proteins containing amino-terminal portions of the intermembrane space protein cytochrome b2 (cyt. b2) fused to dihydrofolate reductase (DHFR) were differentially imported into mitochondria containing mutant hsp70s. A fusion protein between the amino-terminal 167 residues of the precursor of cyt. b2 and DHFR was efficiently transported into mitochondria independently of both hsp70 functions. When the length of the cyt. b2 portion was increased and included the heme binding domain, the fusion protein became dependent on the unfoldase function of mt-hsp70, presumably caused by a conformational restriction of the heme-bound preprotein. In the absence of heme the noncovalent heme binding domain in the longer fusion proteins no longer conferred a dependence on the unfoldase function. When the cyt. b2 portion of the fusion protein was less than 167 residues, its import was still independent of mt-hsp70 function; however, deletion of the intermembrane space sorting signal resulted in preproteins that ended up in the matrix of wild-type mitochondria and whose translocation was strictly dependent on the translocase function of mt-hsp70. These findings provide strong evidence for a dual role of mt-hsp70 in membrane translocation and indicate that preproteins with an intermembrane space sorting signal can be correctly imported even in mutants with severely impaired hsp70 function.

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Characterization of Soluble, Disulfide Bond-Stabilized, Prokaryotically Expressed Human Thyrotropin Receptor Ectodomain*

Endocrinology ◽

10.1210/endo.138.2.4920 ◽

1997 ◽

Vol 138 (2) ◽

pp. 588-593 ◽

Cited By ~ 8

Author(s):

Y. Bobovnikova ◽

P. N. Graves ◽

H. Vlase ◽

T. F. Davies

Keyword(s):

Amino Acids ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Thioredoxin Reductase ◽

Biologically Active ◽

Receptor Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Fusion Partner ◽

E Coli ◽

Recombinant Receptor

Abstract To study the interaction of TSH receptor (TSHR) autoantibodies with receptor protein, it is necessary first to express the receptor in the proper conformation including the formation of correct disulfide bridges. However, the reducing environment of the Escherichia coli (E. coli) cytoplasm prevents the generation of protein disulfide bonds and limits the solubility and immunoreactivity of recombinant human TSHR (hTSHR) products. To circumvent these limitations, hTSHR complementary DNA encoding the extracellular domain (hTSHR-ecd; amino acids 21–415) was inserted into the vector pGEX-2TK by directional cloning and used to transform the thioredoxin reductase mutant strain of E. coli (Ad494), which allowed formation of disulfide bonds in the cytoplasm. After induction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTSHR amino acids 21–35. This showed that over 50% of the expressed hTSHR-ecd was soluble in contrast to expression in a wild-type E. coli (strain αF′), where the majority of the recombinant receptor was insoluble. The soluble recombinant receptor was affinity purified and characterized. Under nonreducing SDS-PAGE conditions, the soluble hTSHR-ecd migrated as refolded, disulfide bond-stabilized, multimeric species, whose formation was independent of fusion partner protein. This product was found to be biologically active as evidenced by the inhibition of the binding of 125I-TSH to the full-length hTSHR expressed in transfected CHO cells and was used to develop a competitive capture enzyme-linked immunosorbent assay for mapping of hTSHR antibody epitopes. Hence, hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation was found to be highly soluble and biologically relevant.

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Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.

The Journal of Cell Biology ◽

10.1083/jcb.127.6.1617 ◽

1994 ◽

Vol 127 (6) ◽

pp. 1617-1626 ◽

Cited By ~ 635

Author(s):

M Furuse ◽

M Itoh ◽

T Hirase ◽

A Nagafuchi ◽

S Yonemura ◽

...

Keyword(s):

Amino Acids ◽

Fusion Protein ◽

Tight Junctions ◽

Integral Membrane Protein ◽

Deletion Mutants ◽

Glutathione S Transferase ◽

E Coli ◽

Vitro Binding ◽

Peripheral Proteins

Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.

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Direct association of pp125FAK with paxillin, the focal adhesion-targeting mechanism of pp125FAK.

Journal of Experimental Medicine ◽

10.1084/jem.182.4.1089 ◽

1995 ◽

Vol 182 (4) ◽

pp. 1089-1099 ◽

Cited By ~ 174

Author(s):

K Tachibana ◽

T Sato ◽

N D'Avirro ◽

C Morimoto

Keyword(s):

Amino Acids ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Fusion Proteins ◽

Focal Adhesions ◽

Immunohistochemical Analysis ◽

Binding Activity ◽

Glutathione S Transferase ◽

Cytoskeleton Protein ◽

Direct Association

Focal adhesion kinase (pp125FAK) is localized to focal adhesions and tyrosine phosphorylated by the engagement of beta 1 integrins. However, it is unclear how pp125FAK is linked to integrin molecules. We demonstrate that pp125FAK is directly associated with paxillin, a 68-kD cytoskeleton protein. The COOH-terminal domain of pp125FAK spanning FAK residues 919-1042 is sufficient for paxillin binding and has vinculin-homologous amino acids, which are essential for paxillin binding. Microinjection and subsequent immunohistochemical analysis reveal that glutathione S-transferase-FAK fusion proteins, which bind to paxillin, localize to focal adhesions, whereas fusion proteins with no paxillin-binding activity do not localize to focal adhesions. These findings strongly suggest that pp125FAK is localized to focal adhesions by the direct association with paxillin.

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Secretion and affinity purification of glutathione S-transferase fusion proteins from yeast

Biotechnology Techniques ◽

10.1007/bf00159408 ◽

1995 ◽

Vol 9 (11) ◽

pp. 821-826

Author(s):

L. A. Castelli ◽

A. J. Petris ◽

S. M. Carroll ◽

I. G. Macreadie

Keyword(s):

Fusion Proteins ◽

Affinity Purification ◽

Glutathione S Transferase

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The primary structure of NG2, a novel membrane-spanning proteoglycan.

The Journal of Cell Biology ◽

10.1083/jcb.114.2.359 ◽

1991 ◽

Vol 114 (2) ◽

pp. 359-371 ◽

Cited By ~ 169

Author(s):

A Nishiyama ◽

K J Dahlin ◽

J T Prince ◽

S R Johnstone ◽

W B Stallcup

Keyword(s):

Amino Acids ◽

Chondroitin Sulfate ◽

Primary Structure ◽

Disulfide Bonds ◽

Transmembrane Domain ◽

Core Protein ◽

Extracellular Domain ◽

Cdna Clones ◽

Amino Terminal ◽

Extracellular Region

The complete primary structure of the core protein of rat NG2, a large, chondroitin sulfate proteoglycan expressed on O2A progenitor cells, has been determined from cDNA clones. These cDNAs hybridize to an mRNA species of 8.9 kbp from rat neural cell lines. The total contiguous cDNA spans 8,071 nucleotides and contains an open reading frame for 2,325 amino acids. The predicted protein is an integral membrane protein with a large extracellular domain (2,224 amino acids), a single transmembrane domain (25 amino acids), and a short cytoplasmic tail (76 amino acids). Based on the deduced amino acid sequence and immunochemical analysis of proteolytic fragments of NG2, the extracellular region can be divided into three domains: an amino terminal cysteine-containing domain which is stabilized by intrachain disulfide bonds, a serine-glycine-containing domain to which chondroitin sulfate chains are attached, and another cysteine-containing domain. Four internal repeats, each consisting of 200 amino acids, are found in the extracellular domain of NG2. These repeats contain a short sequence that resembles the putative Ca(++)-binding region of the cadherins. The sequence of NG2 does not show significant homology with any other known proteins, suggesting that NG2 is a novel species of integral membrane proteoglycan.

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Interaction of MLL Amino Terminal Sequences with Menin Is Required for Transformation.

Blood ◽

10.1182/blood.v106.11.664.664 ◽

2005 ◽

Vol 106 (11) ◽

pp. 664-664

Author(s):

Jay L. Hess ◽

Zhaohai Yang ◽

Haoren Wang ◽

Ya-Xiong Chen ◽

Thomas A. Milne ◽

...

Keyword(s):

Fusion Protein ◽

Rna Polymerase Ii ◽

Fusion Proteins ◽

Target Genes ◽

Hox Genes ◽

Dominant Negative ◽

Similar Distribution ◽

Genetic Ablation ◽

Amino Terminal ◽

Coding Regions

Abstract Rearrangements of the mixed lineage leukemia gene MLL are associated with aggressive lymphoid and myeloid leukemias. The resulting MLL fusion proteins enforce high-level expression of HOX genes including HOX A7 and HOX A9 and the HOX cofactor MEIS1, which is pivotal for leukemogenesis. The mechanism by which this occurs and the relationship to normal MLL function is unknown. MLL and MLL fusion proteins bind with a similar distribution in hematopoietic cells at both promoters and coding sequences of target genes. Our studies suggest that a major mechanism of regulating MLL, which is expressed throughout hematopoiesis, is through modulating it’s binding to target promoters. MLL binds directly to the promoters and coding regions of HOX A7, HOX A9, and MEIS1 only in myeloblasts and not in neutrophils, indicating MLL is physically associated with genes only when they are actively transcribed. Expression of A cluster HOX loci and MEIS1 remains persistently elevated when MLL-ENL or dimerized MLL fusion proteins are expressed. Expression of either fusion protein is associated with increased binding of wild type MLL accompanied by increases in histone acetylation and histone H3 lysine 4, marks that are normally almost completely erased during myeloid differentiation. In addition MLL-ENL induces increased lysine 79 methylation. Both MLL and MLL fusion proteins interact with the tumor suppressor menin via sequences in the extreme amino terminus of MLL. In addition both proteins physically interact with RNA polymerase II, which shows abnormal pausing in the coding regions of HOX genes in Mll null cells. Genetic ablation of menin or expression of a dominant negative inhibitor of the MLL-menin interaction inhibits the growth of MLL fusion protein transformed cells. These findings suggest MLL fusion proteins act in concert with menin, MLL and other coactivators to deregulate HOX gene expression pivotal for transformation.

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Expression in Bacteria of the Gene Encoding the gp43 Antigen of Paracoccidioides brasiliensis: Immunological Reactivity of the Recombinant Fusion Proteins

Clinical and Vaccine Immunology ◽

10.1128/cdli.9.6.1200-1204.2002 ◽

2002 ◽

Vol 9 (6) ◽

pp. 1200-1204 ◽

Cited By ~ 1

Author(s):

Susana N. Diniz ◽

Kátia C. Carvalho ◽

Patrícia S. Cisalpino ◽

José F. Silveira ◽

Luiz R. Travassos ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Fusion Protein ◽

Fusion Proteins ◽

Inclusion Bodies ◽

Paracoccidioides Brasiliensis ◽

Immunological Reactivity ◽

Gene Encoding ◽

Recombinant Fusion Proteins ◽

Gst Fusion Protein

ABSTRACT gp43 is the major diagnostic antigen of Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis (PCM) in humans. In the present study, cDNA of the gp43 gene (PbGP43) was obtained by reverse transcriptase PCR, inserted into a pGEX vector in frame with the glutathione S-transferase (GST) gene, and expressed in Escherichia coli as inclusion bodies. Immunoblotting showed that all sera from patients with chronic pulmonary and acute lymphatic forms of PCM reacted with the recombinant fusion protein of the mature gp43 (381 amino acids). Reactivity with fusion proteins containing subfragments of the N-terminal, internal, or C-terminal regions occurred eventually, and the C-terminal region was the most antigenic. Lack of reactivity with the subfragments may be due to the conformational nature of the gp43 epitopes. Sera from patients with aspergillosis, candidiasis, and histoplasmosis did not react with the gp43-GST fusion protein. Our results suggest that recombinant gp43 corresponding to the processed antigen can be a useful tool in the diagnosis of PCM.

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