scholarly journals Characterization of Human Type X Procollagen and Its NC-1 Domain Expressed as Recombinant Proteins in HEK293 Cells

1998 ◽  
Vol 273 (8) ◽  
pp. 4547-4555 ◽  
Author(s):  
Svenja Frischholz ◽  
Frank Beier ◽  
Irute Girkontaite ◽  
Klaus Wagner ◽  
Ernst Pöschl ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Hek293 Cells ◽  
Human Type

A Single Xyloglucan Xylosyltransferase is Sufficient for Generation of the XXXG Xylosylation Pattern of Xyloglucan

2021 ◽  
Author(s):  
Ruiqin Zhong ◽  
Dennis R Phillips ◽  
Zheng-Hua Ye
Keyword(s):  
Recombinant Proteins ◽  
Cell Walls ◽  
Degree Of Polymerization ◽  
Primary Cell ◽  
Hek293 Cells ◽  
Biochemical Mechanism ◽  
The Third ◽  
Insight Into

Abstarct Xyloglucan is the most abundant hemicellulose in the primary cell walls of dicots. Dicot xyloglucan is the XXXG-type consisting of repeating units of three consecutive xylosylated Glc residues followed by one unsubstituted Glc. Its xylosylation is catalyzed by xyloglucan 6-xylosyltransferases (XXTs) and there exist five XXTs (AtXXT1-5) in Arabidopsis. While AtXXT1and AtXXT2 have been shown to add the first two Xyl residues in the XXXG repeat, which XXTs are responsible for the addition of the third Xyl residue remains elusive although AtXXT5 was a proposed candidate. In this report, we generated recombinant proteins of all five Arabidopsis XXTs and one rice XXT (OsXXT1) in the mammalian HEK293 cells and investigated their ability to sequentially xylosylate Glc residues to generate the XXXG xylosylation pattern. We found that like AtXXT1/2, AtXXT4 and OsXXT1 could efficiently xylosylate the cellohexaose (G6) acceptor to produce mono- and di-xylosylated G6, whereas AtXXT5 was only barely capable of adding one Xyl onto G6. When AtXXT1-catalyzed products were used as acceptors, AtXXT1/2/4 and OsXXT1 but not AtXXT5 were able to xylosylate additional Glc residues to generate tri- and tetra-xylosylated G6. Further characterization of the tri- and tetra-xylosylated G6 revealed that they had the sequence of GXXXGG and GXXXXG with three and four consecutive xylosylated Glc residues, respectively. In addition, we have found that although tri-xylosylation occurred on G6, cello-oligomers with a degree of polymerization of 3 to 5 could only be mono- and di-xylosylated. Together, these results indicate that each of AtXXT1/2/4 and OsXXT1 is capable of sequentially adding Xyl onto three contiguous Glc residues to generate the XXXG xylosylation pattern and these findings provide new insight into the biochemical mechanism underlying xyloglucan biosynthesis.

Molecular characterization of HEK293 cells as emerging versatile cell factories

2021 ◽  
Vol 71 ◽  
pp. 18-24
Author(s):  
Michela Pulix ◽  
Vera Lukashchuk ◽  
Daniel C Smith ◽  
Alan J Dickson
Keyword(s):  
Molecular Characterization ◽  
Hek293 Cells ◽  
Cell Factories

scholarly journals Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alessandro T. Caputo ◽  
Oliver M. Eder ◽  
Hana Bereznakova ◽  
Heleen Pothuis ◽  
Albert Ardevol ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Recombinant Proteins ◽  
Cultured Cells ◽  
Hek293 Cells ◽  
Reaction Products ◽  
Enzyme Form ◽  
X Ray Crystallography ◽  
Mammalian Cell Lines ◽  
Apparent Loss

AbstractPuromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

Characterization of human type II procollagen and collagen-specific antibodies and their application to the study of human type II collagen processing and ultrastructure

1997 ◽  
Vol 16 (1) ◽  
pp. 29-39 ◽  
Author(s):  
Sergio A. Jimenez ◽  
Leena Ala-Kokko ◽  
Darwin J. Prockop ◽  
Carmen F. Merryman ◽  
Nora Shepard ◽  
...  
Keyword(s):  
Type Ii Collagen ◽  
Type Ii ◽  
Specific Antibodies ◽  
Human Type

scholarly journals Characterization of a 190-Kilobase Pair Domain of Human Type I Hair Keratin Genes

1998 ◽  
Vol 273 (41) ◽  
pp. 26683-26691 ◽  
Author(s):  
Michael A. Rogers ◽  
Hermelita Winter ◽  
Christian Wolf ◽  
Marina Heck ◽  
Jürgen Schweizer
Keyword(s):  
Type I ◽  
Human Type ◽  
Hair Keratin

Biophysical and pharmacological characterization of α6-containing nicotinic acetylcholine receptors expressed in HEK293 cells

2014 ◽  
Vol 1542 ◽  
pp. 1-11 ◽  
Author(s):  
Andreas H. Rasmussen ◽  
Dorte Strøbæk ◽  
Tino Dyhring ◽  
Marianne L. Jensen ◽  
Dan Peters ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Hek293 Cells ◽  
Pharmacological Characterization ◽  
Nicotinic Acetylcholine

Characterization of the human type II Na/P i -cotransporter promoter

1998 ◽  
Vol 436 (4) ◽  
pp. 591-598 ◽  
Author(s):  
Helene Hilfiker ◽  
Ivica Kvietikova ◽  
Claudia M. Hartmann ◽  
Gerti Stange ◽  
H. Murer
Keyword(s):  
Type Ii ◽  
Human Type
Sign in / Sign up

Export Citation Format

Share Document