title: Membrane protein expression and production: effects of polyhistidine tag length and position.
abstract: Polyhistidine tags enable the facile purification of proteins by immobilized metal affinity chromatography (IMAC). Both the type and position of purification tags can affect significantly properties of a protein such as its expression level, behavior in solution, and its ability to form suitable samples (esp. suitable crystals for X-ray crystallography). We investigated systematically the effects of polyhistidine tag length and position on many properties related to expression and purification of recombinant integral membrane proteins. Specifically, modified Escherichia coli pET expression vectors were built that placed 6- or 10-histidine tags at the N- or C-termini of the subcloned gene. The E. coli water channel AqpZ was subcloned into this suite of vectors and its expression, purification, solution properties, and yield were characterized. These studies show that: (1) all vectors yield similar expression levels, (2) tag length has a greater effect than tag position upon yield, (3) neither tag length nor position affects significantly detergent solubilization of the protein, (4) the length of the tag affects the oligomerization state of the purified protein, and (5) the tag length and position change chromatographic behavior of the detergent-solubilized protein. In addition, substitution of the lysine codon AAA at the second position, previously shown to have some effect upon soluble protein expression levels, did not have a large effect on AqpZ production. We are currently producing approximately 12 mg of purified AqpZ per liter of shake-flask culture, and preliminary crystals that diffract to approximately 5A resolution have been obtained.
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908-0736, USA.
authors.forename: Arun K
authors.initials: AK
authors.lastname: Mohanty
authors.forename: Michael C
authors.initials: MC
authors.lastname: Wiener
mesh_terms: D020346:Aquaporins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility
chemical_list: D020346:Aquaporins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009841:Oligonucleotides; D011994:Recombinant Proteins; C106425:aqpZ protein, E coli; C033223:polyhistidine; D006639:Histidine