title: A PCR-directed cell-free approach to optimize protein expression using diverse fusion tags.
abstract: N-terminal fusion tags that enhance translation initiation or protein solubility are often used to facilitate protein overexpression. As the optimal tag for a given target protein cannot be predicted a priori, valuable time can be lost in cloning and manipulating the corresponding gene to generate different fusion constructs for expression analysis. We have developed a cell-free strategy that consolidates these steps, enabling the utility of a panel of nine fusion-tags to be determined within one to two days. This approach exploits the fact that PCR-amplified DNA can be used as a template for cell-free protein synthesis. Overlap/extension PCR using the TEV protease site as the overlap region allows the fusion of different T7 promoter (T7p)-tag-TEV DNA fragments with a TEV-gene-T7 terminator (T7ter) fragment. For tag sequences where the TEV site is not compatible, a short C₃G₃ repeat (CGr) sequence can be used as the overlap region. The resulting T7p-tag-TEV-gene-T7ter constructs are then used as templates for PCR-directed cell-free protein synthesis to identify which tag-TEV-gene fusion protein produces the highest amount of soluble protein. We have successfully applied this approach to the overexpression of the Adiponectin hypervariable domain (AHD). Five of the nine N-terminal fusion tags tested enabled the synthesis of soluble recombinant protein. The best of these was the Peptidyl-prolylcis-trans isomerise B (PpiB) fusion tag which produces 1mg/ml amounts of soluble fusion protein. PpiB is an example of a new class of fusion tag known as the "stress-responsive proteins". Our results suggest that this cell-free fusion-tag expression screen facilitates the rapid identification of suitable fusion-tags that overcome issues such as poor expression and insolubility, often encountered using conventional approaches.
authors: The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland, New Zealand. Andrew.Kralicek@plantandfood.co.nz
authors: Andrew V
authors: AV
authors: Kralicek
authors: Mazdak
authors: M
authors: Radjainia
authors: Nazratul A B
authors: NA
authors: Mohamad Ali
authors: Colm
authors: C
authors: Carraher
authors: Richard D
authors: RD
authors: Newcomb
authors: Alok K
authors: AK
authors: Mitra
mesh_terms: D052242:Adiponectin / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D010450:Endopeptidases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics*
chemical_list: D052242:Adiponectin; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D010450:Endopeptidases; C099488:TEV protease