Optimized functional and structural design of dual-target LMRAP, a bifunctional fusion protein with a 25-amino-acid antitumor peptide and GnRH Fc fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

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Acquisition of NFKB1-selective DNA binding by substitution of four amino acid residues from NFKB1 into RelA

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3850-3859.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3850-3859

Author(s):

T A Coleman ◽

C Kunsch ◽

M Maher ◽

S M Ruben ◽

C A Rosen

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Fusion Protein ◽

Binding Specificity ◽

Single Amino Acid ◽

Nf Kappa B ◽

Dna Binding Specificity ◽

Dna Motif ◽

Single Amino Acid Change

The subunits of NF-kappa B, NFKB1 (formerly p50) and RelA (formerly p65), belong to a growing family of transcription factors that share extensive similarity to the c-rel proto-oncogene product. The homology extends over a highly conserved stretch of approximately 300 amino acids termed the Rel homology domain (RHD). This region has been shown to be involved in both multimerization (homo- and heterodimerization) and DNA binding. It is now generally accepted that homodimers of either subunit are capable of binding DNA that contains a kappa B site originally identified in the immunoglobulin enhancer. Recent studies have demonstrated that the individual subunits of the NF-kappa B transcription factor complex can be distinguished by their ability to bind distinct DNA sequence motifs. By using NFKB1 and RelA subunit fusion proteins, different regions within the RHD were found to confer DNA-binding and multimerization functions. A fusion protein that contains 34 N-terminal amino acids of NFKB1 and 264 amino acids of RelA displayed preferential binding to an NFKB1-selective DNA motif while dimerizing with the characteristics of RelA. Within the NFKB1 portion of this fusion protein, a single amino acid change of His to Arg altered the DNA-binding specificity to favor interaction with the RelA-selective DNA motif. Furthermore, substitution of four amino acids from NFKB1 into RelA was able to alter the DNA-binding specificity of the RelA protein to favor interaction with the NFKB1-selective site. Taken together, these findings demonstrate the presence of a distinct subdomain within the RHD involved in conferring the DNA-binding specificity of the Rel family of proteins.

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In Vivo Marking of Spontaneous or Vaccine-Induced Fibrosarcomas in the Domestic House Cat, Using an Adenoviral Vector Containing a Bifunctional Fusion Protein, GAL-TEK

Human Gene Therapy ◽

10.1089/hum.1995.6.9-1215 ◽

1995 ◽

Vol 6 (9) ◽

pp. 1215-1223 ◽

Cited By ~ 8

Author(s):

Frank C. Marini Iii ◽

John P. Cannon ◽

John W. Belmont ◽

Edward J. Shillitoe ◽

Jean-Numa Lapeyre

Keyword(s):

Fusion Protein ◽

Adenoviral Vector ◽

Bifunctional Fusion Protein

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SPECIFICITY IN THE COMBINATION OF FD FRAGMENTS WITH L CHAINS TO FORM HAPTEN-BINDING SITES

Journal of Experimental Medicine ◽

10.1084/jem.123.5.921 ◽

1966 ◽

Vol 123 (5) ◽

pp. 921-934 ◽

Cited By ~ 12

Author(s):

O. A. Roholt ◽

G. Radzimski ◽

D. Pressman

Keyword(s):

Amino Acid ◽

Propionic Acid ◽

Binding Sites ◽

Amino Acid Analysis ◽

Binding Activity ◽

Light Chains ◽

Antibody Activity ◽

Fc Fragment ◽

Fab Fragments ◽

Correct Alignment

In the work reported here we have shown that light chains and Fd fragments can be separated completely in propionic acid and then recombined to form Fab fragments with antibody activity. This experiment indicates that in the recombination a correct alignment of the Fd fragments and the L chains occurs to give a competent antibody site, just as occurs with the recombination of separated heavy and light chains of the antibody; thus the Fc fragment is not required for correct alignment. Fd fragments of antibody alone show very low binding activity toward the specific hapten. As is the case for the combination of heavy and light chains, the combination of Fd fragments and light chains also requires that both components come from antibody from the same rabbit in order to give binding sites. When they are derived from different rabbits producing antibody against the same antigen, they still give Fab fragments as shown by immunoelectrophoresis but do not have competent binding sites. An important observation is that the subunits of the papain digest fractions, FabI and FabII, have the capacity to cross-combine to form active Fab fragments with competent binding sites. FdI from FabI combines with LII chains from FabII to give the composite (FdI-LII) with good binding activity. Likewise, the composite (FdII-LI) has good binding activity. The composites from the two types of antibody molecules yielding different Fab fragments have antibody activity although heretofore these molecules have appeared to be different on the bases of chromatography and amino acid analysis. There is also a preferential combination of the Fd fragments to combine with the correct L fragments to give binding sites since this combination takes preference over the combination of Fd fragments of antibody with light chains of normal globulin (or of light chains of antibody with Fd fragments of normal globulin).

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Sequence Analysis and Expression of the Attachment and Fusion Proteins of Canine Distemper Virus Wild-Type Strain A75/17

Journal of Virology ◽

10.1128/jvi.73.3.2263-2269.1999 ◽

1999 ◽

Vol 73 (3) ◽

pp. 2263-2269 ◽

Cited By ~ 31

Author(s):

Pascal Cherpillod ◽

Karin Beck ◽

Andreas Zurbriggen ◽

Riccardo Wittek

Keyword(s):

Amino Acid ◽

Fusion Protein ◽

Translation Initiation ◽

Fusion Proteins ◽

Canine Distemper Virus ◽

Protein A ◽

Biological Properties ◽

Canine Distemper ◽

Wild Type ◽

Attachment Protein

ABSTRACT The biological properties of wild-type A75/17 and cell culture-adapted Onderstepoort canine distemper virus differ markedly. To learn more about the molecular basis for these differences, we have isolated and sequenced the protein-coding regions of the attachment and fusion proteins of wild-type canine distemper virus strain A75/17. In the attachment protein, a total of 57 amino acid differences were observed between the Onderstepoort strain and strain A75/17, and these were distributed evenly over the entire protein. Interestingly, the attachment protein of strain A75/17 contained an extension of three amino acids at the C terminus. Expression studies showed that the attachment protein of strain A75/17 had a higher apparent molecular mass than the attachment protein of the Onderstepoort strain, in both the presence and absence of tunicamycin. In the fusion protein, 60 amino acid differences were observed between the two strains, of which 44 were clustered in the much smaller F2 portion of the molecule. Significantly, the AUG that has been proposed as a translation initiation codon in the Onderstepoort strain is an AUA codon in strain A75/17. Detailed mutation analyses showed that both the first and second AUGs of strain A75/17 are the major translation initiation sites of the fusion protein. Similar analyses demonstrated that, also in the Onderstepoort strain, the first two AUGs are the translation initiation codons which contribute most to the generation of precursor molecules yielding the mature form of the fusion protein.

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Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens.

Acta Biochimica Polonica ◽

10.18388/abp.2003_3736 ◽

2003 ◽

Vol 50 (1) ◽

pp. 269-278

Author(s):

Amr M Shabaan ◽

Magdy M Mohamed ◽

Mohga S Abdallah ◽

Hayat M Ibrahim ◽

Amr M Karim

Keyword(s):

Amino Acid ◽

Fusion Protein ◽

Schistosoma Mansoni ◽

Molecular Mass ◽

Vaccine Development ◽

Complete Sequence ◽

Cdna Clones ◽

Western Blots ◽

Calculated Molecular Mass ◽

Reading Frame

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.

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The two major membrane skeletal proteins (articulins) of Euglena gracilis define a novel class of cytoskeletal proteins.

The Journal of Cell Biology ◽

10.1083/jcb.118.6.1465 ◽

1992 ◽

Vol 118 (6) ◽

pp. 1465-1475 ◽

Cited By ~ 45

Author(s):

J A Marrs ◽

G B Bouck

Keyword(s):

Amino Acid ◽

Fusion Protein ◽

Euglena Gracilis ◽

Cytoskeletal Proteins ◽

Cdna Clones ◽

Thin Sections ◽

Core Domain ◽

The Core ◽

Amino Acid Repeats ◽

Peptide Maps

60% of the peripheral membrane skeleton of Euglena gracilis consists of equimolar amounts of two proteins (articulins) with M(r)s in SDS gels of 80 and 86 kD. To understand eventually how these proteins assemble and function in maintaining cell form and membrane integrity we have undertaken a molecular characterization of articulins. A lambda gt11 expression library constructed from Euglena gracilis mRNAs was screened with antibodies against both articulins. Two sets of cDNAs were recovered, and evidence from three independent assays confirmed that both sets encoded articulins: (a) Anti-articulin antibodies recognized a high molecular weight beta-galactosidase (beta-gal) fusion protein expressed in bacteria infected with lambda gt11 cDNA clones. (b) Antibodies generated against the bacterially expressed beta-gal fusion protein identified one or the other articulin in Western blots of Euglena proteins. These antibodies also localized to the membrane skeletal region in thin sections of Euglena. (c) Peptide maps of the beta-gal fusion protein were similar to peptide maps of Euglena articulins. From the nucleotide sequence of the two sets of cDNAs an open reading frame for each articulin was deduced. In addition to 37% amino acid identity and overall structural similarity, both articulins exhibited a long core domain consisting of over 30 12-amino acid repeats with the consensus VPVPV--V--. Homology plots comparing the same or different articulins revealed larger, less regular repeats in the core domain that coincided with predicted turns in extended beta-sheets. Outside the core domain a short hydrophobic region containing four seven-amino acid repeats (consensus: APVTYGA) was identified near the carboxy terminus of the 80-kD articulin, but near the amino terminus of the 86-kD articulin. No extensive sequence similarities were found between articulins and other protein sequences in various databanks. We conclude that the two articulins are related members of a new class of membrane cytoskeletal proteins.

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