Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells

Research output: Contribution to journal › Journal article › Research › peer-review

Standard

Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells. / Hunter, Roger W.; Zeqiraj, Elton; Morrice, Nicholas; Sicheri, Frank; Sakamoto, Kei.

In: Protein Expression and Purification, Vol. 108, 04.2015, p. 23-29.

Research output: Contribution to journal › Journal article › Research › peer-review

Harvard

Hunter, RW, Zeqiraj, E, Morrice, N, Sicheri, F & Sakamoto, K 2015, 'Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells', Protein Expression and Purification, vol. 108, pp. 23-29. https://doi.org/10.1016/j.pep.2014.12.007

APA

Hunter, R. W., Zeqiraj, E., Morrice, N., Sicheri, F., & Sakamoto, K. (2015). Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells. Protein Expression and Purification, 108, 23-29. https://doi.org/10.1016/j.pep.2014.12.007

Vancouver

Hunter RW, Zeqiraj E, Morrice N, Sicheri F, Sakamoto K. Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells. Protein Expression and Purification. 2015 Apr;108:23-29. https://doi.org/10.1016/j.pep.2014.12.007

Author

Hunter, Roger W. ; Zeqiraj, Elton ; Morrice, Nicholas ; Sicheri, Frank ; Sakamoto, Kei. / Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells. In: Protein Expression and Purification. 2015 ; Vol. 108. pp. 23-29.

Bibtex

@article{ce3fb3c21e0f48f5ad4f8b269c07b866,
title = "Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells",
abstract = "We report the successful expression and purification of functional human muscle glycogen synthase (GYS1) in complex with human glycogenin-1 (GN1). Stoichiometric GYS1:GN1 complex was produced by co-expression of GYS1 and GN1 using a bicistronic pFastBac{\texttrademark}-Dual expression vector, followed by affinity purification and subsequent size-exclusion chromatography. Mass spectrometry analysis identified that GYS1 is phosphorylated at several well-characterised and uncharacterised Ser/Thr residues. Biochemical analysis, including activity ratio (in the absence relative to that in the presence of glucose-6-phosphate) measurement, covalently attached phosphate estimation as well as phosphatase treatment, revealed that recombinant GYS1 is substantially more heavily phosphorylated than would be observed in intact human or rodent muscle tissues. A large quantity of highly-pure stoichiometric GYS1:GN1 complex will be useful to study its structural and biochemical properties in the future, which would reveal mechanistic insights into its functional role in glycogen biosynthesis.",
keywords = "Energy metabolism, Glucosyltransferase, Glycogenesis, Phosphorylation",
author = "Hunter, {Roger W.} and Elton Zeqiraj and Nicholas Morrice and Frank Sicheri and Kei Sakamoto",
year = "2015",
month = apr,
doi = "10.1016/j.pep.2014.12.007",
language = "English",
volume = "108",
pages = "23--29",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press",

}

RIS

TY - JOUR

T1 - Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells

AU - Hunter, Roger W.

AU - Zeqiraj, Elton

AU - Morrice, Nicholas

AU - Sicheri, Frank

AU - Sakamoto, Kei

PY - 2015/4

Y1 - 2015/4

N2 - We report the successful expression and purification of functional human muscle glycogen synthase (GYS1) in complex with human glycogenin-1 (GN1). Stoichiometric GYS1:GN1 complex was produced by co-expression of GYS1 and GN1 using a bicistronic pFastBac™-Dual expression vector, followed by affinity purification and subsequent size-exclusion chromatography. Mass spectrometry analysis identified that GYS1 is phosphorylated at several well-characterised and uncharacterised Ser/Thr residues. Biochemical analysis, including activity ratio (in the absence relative to that in the presence of glucose-6-phosphate) measurement, covalently attached phosphate estimation as well as phosphatase treatment, revealed that recombinant GYS1 is substantially more heavily phosphorylated than would be observed in intact human or rodent muscle tissues. A large quantity of highly-pure stoichiometric GYS1:GN1 complex will be useful to study its structural and biochemical properties in the future, which would reveal mechanistic insights into its functional role in glycogen biosynthesis.

AB - We report the successful expression and purification of functional human muscle glycogen synthase (GYS1) in complex with human glycogenin-1 (GN1). Stoichiometric GYS1:GN1 complex was produced by co-expression of GYS1 and GN1 using a bicistronic pFastBac™-Dual expression vector, followed by affinity purification and subsequent size-exclusion chromatography. Mass spectrometry analysis identified that GYS1 is phosphorylated at several well-characterised and uncharacterised Ser/Thr residues. Biochemical analysis, including activity ratio (in the absence relative to that in the presence of glucose-6-phosphate) measurement, covalently attached phosphate estimation as well as phosphatase treatment, revealed that recombinant GYS1 is substantially more heavily phosphorylated than would be observed in intact human or rodent muscle tissues. A large quantity of highly-pure stoichiometric GYS1:GN1 complex will be useful to study its structural and biochemical properties in the future, which would reveal mechanistic insights into its functional role in glycogen biosynthesis.

KW - Energy metabolism

KW - Glucosyltransferase

KW - Glycogenesis

KW - Phosphorylation

UR - http://www.scopus.com/inward/record.url?scp=84922691744&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2014.12.007

DO - 10.1016/j.pep.2014.12.007

M3 - Journal article

C2 - 25527037

AN - SCOPUS:84922691744

VL - 108

SP - 23

EP - 29

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

ER -

ID: 239212865