Preparing mFc- and hFc-Fusion Proteins from Mammalian Cells

2021 ◽  
Vol 2021 (8) ◽  
pp. pdb.prot100040
Author(s):  
Edward A. Greenfield ◽  
James DeCaprio ◽  
Mohan Brahmandam
Keyword(s):  
Fusion Proteins ◽  
Mammalian Cells

scholarly journals Expression of Hirudin Fusion Proteins in Mammalian Cells

Circulation ◽  
1998 ◽  
Vol 98 (24) ◽  
pp. 2744-2752 ◽  
Author(s):  
Kristian Riesbeck ◽  
Daxin Chen ◽  
Geoffrey Kemball-Cook ◽  
John H. McVey ◽  
Andrew J. T. George ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Mammalian Cells

scholarly journals Transcriptional repression by Drosophila and mammalian Polycomb group proteins in transfected mammalian cells.

1994 ◽  
Vol 14 (3) ◽  
pp. 1721-1732 ◽  
Author(s):  
C A Bunker ◽  
R E Kingston
Keyword(s):  
Binding Sites ◽  
Transcriptional Repression ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Polycomb Group ◽  
Polycomb Group Proteins ◽  
Related Gene ◽  
Activation Domains ◽  
Sex Combs ◽  
Significant Homology

The Polycomb group (Pc-G) genes are essential for maintaining the proper spatially restricted expression pattern of the homeotic loci during Drosophila development. The Pc-G proteins appear to function at target loci to maintain a state of transcriptional repression. The murine oncogene bmi-1 has significant homology to the Pc-G gene Posterior sex combs (Psc) and a highly related gene, Suppressor two of zeste [Su(z)2]. We show here that the proteins encoded by bmi-1 and the Pc-G genes Polycomb (Pc) and Psc as well as Su(z)2 mediate repression in mammalian cells when targeted to a promoter by LexA in a cotransfection system. These fusion proteins repress activator function by as much as 30-fold, and the effect on different activation domains is distinct for each Pc-G protein. Repression is observed when the LexA fusion proteins are bound directly adjacent to activator binding sites and also when bound 1,700 bases from the promoter. These data demonstrate that the products of the Pc-G genes can significantly repress activator function on transiently introduced DNA. We suggest that this function contributes to the stable repression of targeted loci during development.

scholarly journals Generation of antisera that discriminate among mammalian alpha-tubulins: introduction of specialized isotypes into cultured cells results in their coassembly without disruption of normal microtubule function.

1988 ◽  
Vol 106 (6) ◽  
pp. 2011-2022 ◽  
Author(s):  
W Gu ◽  
S A Lewis ◽  
N J Cowan
Keyword(s):  
Immune Response ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Alpha Tubulin ◽  
Tubulin Isotypes ◽  
Tubulin Isotype ◽  
Carboxy Terminal ◽  
Nih 3T3 ◽  
Shared Epitopes

To assay the functional significance of the multiple but closely related alpha-tubulin polypeptides that are expressed in mammalian cells, we generated three specific immune sera, each of which uniquely recognizes a distinct alpha-tubulin isotype. All three isotypes are expressed in a tissue-restricted manner: one (M alpha 3/7) only in mature testis, one (M alpha 4) mainly in muscle and brain, and the third (M alpha 6) in several tissues at a very low level. A fourth specific antiserum was also generated that distinguishes between the tyrosinated and nontyrosinated form of a single alpha-tubulin isotype. Because individual tubulin isotypes cannot be purified biochemically, these sera were raised using cloned fusion proteins purified from host Escherichia coli cells. To suppress the immune response to shared epitopes, animals were first rendered tolerant to fusion proteins encoding all but one of the known mammalian alpha-tubulin isotypes. Subsequent challenge with the remaining fusion protein then resulted in the elicitation of an immune response to unique epitopes. Three criteria were used to establish the specificity of the resulting sera: (a) their ability to discriminate among cloned fusion proteins representing all the known mammalian alpha-tubulin isotypes; (b) their ability to uniquely detect alpha-tubulin in whole extracts of tissues; and (c) their capacity to stain microtubules in fixed preparations of cells transfected with sequences encoding the corresponding isotype. The transfection experiments served to demonstrate (a) the coassembly of M alpha 3/7, M alpha 4, and M alpha 6 into both interphase and spindle microtubules in HeLa cells and NIH 3T3 cells, and (b) that the M alpha 4 isotype, which is unique among mammalian alpha-tubulins in that it lacks an encoded carboxy-terminal tyrosine residue, behaves like other alpha-tubulin isotypes with respect to the cycle of tyrosination/detyrosination that occurs in most cultured cells.

Single-Stranded DNA-Binding Proteins (SSBPs) Promote the Oligomerization of LIM-Domain Binding Protein Ldb1

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2437-2437
Author(s):  
Ying Cai ◽  
Lalitha Nagarajan ◽  
Stephen J. Brandt
Keyword(s):  
Dna Binding ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Binding Activity ◽  
Luciferase Reporter ◽  
Lim Domain ◽  
Single Stranded Dna ◽  
Dna Binding Activity ◽  
E Box

Abstract The multifunctional LIM domain-binding protein Ldb1 is important in multiple developmental programs, including hematopoiesis. An evolutionarily conserved family of proteins with single-stranded DNA-binding activity, the SSBPs, has been shown to act as Ldb1 partners and augment its biological actions. We recently established that Ssbp2 and Ssbp3 were components of an E-box-GATA DNA-binding complex in murine erythroid progenitors containing the LIM-only protein Lmo2 and transcription factors Tal1, E2A, and Gata1 and showed these SSBPs stimulated E box-GATA DNA-binding activity and inhibited Ldb1 ubiquitination and subsequent proteasomal degradation (Genes & Dev.21:942–955, 2007). As its SSBP interaction domain (Ldb1/Chip conserved domain or LCCD) is adjacent to Ldb1’s N-terminal dimerization domain (DD), we sought to determine whether SSBP binding affected Ldb1 dimerization. To investigate, the Ldb1 coding region was fused to the DNA-binding domain of the yeast transcription factor GAL4 (GAL4DBD) and in a second construct to the activation domain of herpesvirus VP16 (VP16AD). These fusion proteins were then expressed in mammalian cells with a luciferase reporter linked to a promoter with iterated GAL4 binding sites. Luciferase activity became detectable with coexpression of the VP16AD-Ldb1 and GAL4DBD-Ldb1 fusions, presumably from Ldb1 dimerization, which increased markedly with simultaneous expression of SSBP2. In contrast, SSBP2 (ΔLUFS) and Ldb1 (ΔLCCD) mutants incapable of interacting with Ldb1 and SSBPs, respectively, were inactive, suggesting that SSBP2 augmentation of Ldb1 dimerization involved direct protein-protein interactions. To exclude an effect of SSBP2 on turnover of Ldb1 fusion proteins, radiolabeled full-length Ldb1 and SSBP3 were prepared by in vitro transcription/translation, mixed, and subjected to chemical crosslinking. Addition of the crosslinker bis(sulfosuccinimidyl)-suberate (BS3) to Ldb1, but not SSBP3, led to the appearance of a radiolabeled protein with mobility in denaturing polyacrylamide gels approximately twice that of Ldb1, consistent with an Ldb1 homodimer. When SSBP3 and Ldb1 were mixed together and crosslinked, a dose-related increase was noted in a more retarded species predicted to contain two molecules each of Ldb1 and SSBP3, together with a decrease in monomeric Ldb1. Finally, two well-characterized dimerization-defective Ldb1 mutants, Ldb1(200–375) and Ldb1(50–375), failed to support the formation of the higher molecular weight species or to homodimerize. Thus, the SSBPs promoted assembly of ternary complexes incorporating both SSBP and Ldb1 in a manner dependent on Ldb1 dimerization. The failure to observe Ldb1-SSBP heterodimers in cross-linking experiments suggests, further, that the SSBPs interacted with preformed Ldb1 dimers. In summary, either through an allosteric effect on Ldb1’s DD or by altering the equilibrium between monomeric and dimeric species, the SSBPs promote Ldb1 oligomerization. Together with inhibition of Ldb1 ubiquitination and turnover, this would serve to augment Ldb1 function.

scholarly journals Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Sanum Bashir ◽  
Tu Dang ◽  
Jana Rossius ◽  
Johanna Wolf ◽  
Ralf Kühn
Keyword(s):  
Fusion Proteins ◽  
Mammalian Cells ◽  
Gene Editing ◽  
Hek293 Cells ◽  
Histone H2a ◽  
Traffic Light ◽  
Reporter System ◽  
Binding Domains ◽  
And Shifts ◽  
Endogenous Loci

Abstract Background Precise genetic modifications are preferred products of CRISPR-Cas9 mediated gene editing in mammalian cells but require the repair of induced double-strand breaks (DSB) through homology directed repair (HDR). Since HDR competes with the prevailing non-homologous end joining (NHEJ) pathway and depends on the presence of repair templates its efficiency is often limited and demands optimized methodology. Results For the enhancement of HDR we redirect the DSB repair pathway choice by targeting the Ubiquitin mark for damaged chromatin at Histone H2A-K15. We used fusions of the Ubiquitin binding domain (UBD) of Rad18 or RNF169 with BRCA1 to promote HDR initiation and UBD fusions with DNA binding domains to attract donor templates and facilitate HDR processing. Using a traffic light reporter system in human HEK293 cells we found that the coexpression of both types of UBD fusion proteins promotes HDR, reduces NHEJ and shifts the HDR/NHEJ balance up to 6-fold. The HDR enhancing effect of UBD fusion proteins was confirmed at multiple endogenous loci. Conclusions Our findings provide a novel efficient approach to promote precise gene editing in human cells.

Novel methods to produce fusion proteins in mammalian cells: fusions in DNA, RNA and protein levels

2009 ◽  
Vol 108 ◽  
pp. S96
Author(s):  
Hiroshi Ueda ◽  
Ryohei Iwasaki ◽  
Akihito Tanabe ◽  
Chung-Yong Han ◽  
Hiroki Kiuchi ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Mammalian Cells ◽  
Protein Levels ◽  
Novel Methods

Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration

10.1038/4042 ◽  
1998 ◽  
Vol 4 (12) ◽  
pp. 1449-1452 ◽  
Author(s):  
Hikaru Nagahara ◽  
Adamina M. Vocero-Akbani ◽  
Eric L. Snyder ◽  
Alan Ho ◽  
Dawn G. Latham ◽  
...  
Keyword(s):  
Cell Migration ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Full Length ◽  
Tat Fusion Proteins
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