Construction, expression, and purification of recombinant αVβ5 integrin

Abstract

A recombinant integrin expression system has been created for the large-scale production of αVβ5 integrin extracellular domains that take advantage of Fos and Jun dimerization for expression in bacterial, insect, and mammalian cells. This utilizes an all-in-one vector, pQE-TriSystem, with molecular machinery for parallel expression without the need of additional subcloning. Optimal expression in HEK293 cells was determined by a time course analysis. The heterodimer was purified in a one-step nickel column purification scheme, and the sequence and functional state were confirmed by mass spectrometry and inhibition assays, respectively. The yields of αVβ5 integrin obtained are in quantities suitable for multiple applications including structural biology and functional assays.

Expression, purification and renaturation of truncated human integrin β1 from inclusion bodies of Escherichia coli

“…The productivity was higher than that expressed by eukaryotes. Tartaglia et al overexpress integrin aVb5 using HEK293 cells and obtained a yield about 2 mg per cell factory (equivalent to forty 15 cm plates) [14], however the molecular weight of ITGB1-head described here was much less than their protein.…”

“…There have been a few studies focusing on the expression of the extracelluar region of integrin b1 [14], and to the best of our knowledge, none of them utilized a prokaryotic expression system. In present study, an efficient process was shown to produce highly purified, soluble and active integrin b1 ectodomain using Escherichia coli.…”

“…How these two conformations switch is still unknown although research suggests that the relationship between the ligands and integrin activation [12], or the signal transduction involved in this process [13] are critical. Being able to obtain enough purified integrin b1 protein is a prerequisite to carrying out these types of studies, especially for in vitro experiments.There have been a few studies focusing on the expression of the extracelluar region of integrin b1 [14], and to the best of our knowledge, none of them utilized a prokaryotic expression system. In present study, an efficient process was shown to produce highly purified, soluble and active integrin b1 ectodomain using Escherichia coli.…”

Molecular cloning, overexpression, and an efficient one-step purification of α5β1 integrin

“…2B and C). The completed constructs are similar to those previously published for the expression of αV-Fos and β5-Jun with the exception that they utilized the pQE-TriVector System [21]. However, overlap between the α5 cDNA and Nco I restriction digest sites within the pQE-TriVector precluded its use for cloning/expression in the current study.…”