Membrane Proteins - Production and Functional Characterization (eBook)

Arun K. Shukla (Herausgeber)

eBook Download: PDF | EPUB

2015 | 1. Auflage
670 Seiten
Elsevier Science (Verlag)
978-0-12-801625-1 (ISBN)

Infos

Membrane Proteins - Production and Function Characterization a volume of Methods in Enzymology, encompasses chapters from the leading experts in the area of membrane protein biology. The chapters provide a brief overview of the topics covered and also outline step-by-step protocol. Illustrations and case example images are included wherever appropriate to help the readers understand the schematics and general experimental outlines.

• Volume of Methods In Enzymology
• Contains a collection of a diverse array of topics in the area of membrane protein biology ranging from recombinant expression, isolation, functional characterization, biophysical studies and crystallization

Textauszug

Chapter One

Engineering Escherichia coli for Functional Expression of Membrane Proteins

Franz Y. Ho; Bert Poolman1    Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute & Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands
1 Corresponding author: email address: [e-mail]

Abstract

A major bottleneck in the characterization of membrane proteins is low yield of functional protein in recombinant expression. Microorganisms are widely used for recombinant protein production, because of ease of cultivation and high protein yield. However, the target proteins do not always obtain their native conformation and may end up in a nonfunctional state, in insoluble aggregates. For screening of functional protein, it is thus important to readily discriminate aggregated, mistargeted protein from globally well-folded, membrane-inserted protein. We developed a robust strategy for expression screening of functional proteins in bacteria, which is based on directed evolution. In this strategy, the C-terminus of the target membrane protein is tagged with two additional protein domains in tandem. The first one is green fluorescent protein (GFP), which functions as a reporter of the global folding state of the fusion protein. The other one is the erythromycin resistance protein (23S ribosomal RNA adenine N-6 methyltransferase, ErmC), which confers a means to select for enhanced expression. By gradually increasing the antibiotic concentration in the medium, we force the cells to evolve in a way that allows more functional target-GFP–ErmC to be expressed. The acquired genomic mutations can be generic or membrane protein specific. This strategy is readily adopted for the expression of any protein and ultimately yields a wealth of genomic data that may provide insight into the factors that limit the production of given classes or types of proteins.

Keywords

Directed evolution

Strain engineering

Protein expression

Folding reporter

Membrane proteins

1 Introduction

Integral membrane proteins contribute 15–30% of the open-reading frames found in the genomes of organisms from all domains of life (Bendtsen, Binnewies, Hallin, & Ussery, 2005; Wallin & von Heijne, 1998). However, our understanding of function–structure relationships of membrane proteins is low compared to water-soluble proteins (Granseth, Seppälä, Rapp, Daley, & Von Heijne, 2007). There are many difficulties in studying membrane proteins, and the challenge already starts with the expression of the genes and production of the proteins in a functional state. Recombinant expression in heterologous hosts is the most versatile strategy, but difficult to produce proteins often cause toxicity to the cells and end up themselves in insoluble aggregates (Bill et al., 2011). Expression conditions can be optimized but this is a laborious process (Francis & Page, 2010).

Large-scale production of proteins is commonly achieved using microorganisms, because of the ease of growth and many genetic and biochemical tools are available. Several factors determine the production level and functionality of the proteins, such as (i) the toxicity the proteins evoke to the host cells (Wagner et al., 2007), (ii) the posttranslational modifications required for structure and/or functionality (Grisshammer & Tate, 1995), (iii) the difference in codon usage between the recombinant gene and the expression host (Angov, Hillier, Kincaid, & Lyon, 2008), (iv) limitations of protein synthesis precursors such as tRNA and amino acids (Marreddy et al., 2010; Puri et al., 2014), (v) overloading of foldase and chaperone activities (Tate, Whiteley, & Betenbaugh, 1999), (vi) saturation of the membrane protein insertion machinery (Loll, 2003; Wagner, Bader, Drew, & de Gier, 2006), and (vii) uncoordinated protein biosynthesis kinetics (Bill et al., 2011; De Marco, 2013). Membrane proteins have a complex biogenesis pathway, requiring chaperones and machineries for membrane insertion, protein folding, and oligomerization, but often the limiting factor(s) leading to low or nonfunctional expression is not understood. Rather than screening numbers of expression conditions, it is easier to improve the production by evolving the host.

In this method, we describe a simple procedure for engineering Escherichia coli by forcing the cells to produce more desired protein by fusing a folding indicator and antibiotic resistance marker to its C-terminus (Fig. 1A). The same strategy has been proven successful for the overexpression of membrane proteins in both the Gram-positive bacterium Lactococcus lactis and the Gram-negative bacterium E. coli (Gul, Linares, Ho, & Poolman, 2014; Linares, Geertsma, & Poolman, 2010).

Figure 1 (A) Cartoon of fusion protein used for strain selection. The folding reporter GFP and the selection marker encoding the 23S ribosomal RNA adenine N-6-methyltransferase (ErmC) are fused in tandem at the C-terminus of the target membrane protein. N and C indicate the N- and C-terminus of the fusion protein. (B) Transport activity of LacSΔIIA fused with GFP in E. coli MC1061 cells. The expression of LacSΔIIA was controlled by the concentration of l-arabinose. (C) Expression of LacSΔIIA–GFP fusion was analyzed by SDS-PAGE. Upper panel: in-gel fluorescence; lower panel: anti-His immunoblot. White and black arrows indicate the folded and misfolded fusion protein, respectively. (D) Confocal microscopy images of E. coli MC1061 cells expressing LacSΔIIA–GFP to different levels; the l-arabinose concentration is indicated above the panels. Upper panel: close-up of cells to indicate the distribution of the fluorescence (scale bar, 2 μm). Lower panel: overview of the culture (scale bar, 10 μm). Panels (B–D) are modified with permission from Geertsma, Groeneveld, Slotboom, and Poolman (2008); copyright (2008) by the National Academy of Sciences USA.

1.1 Green fluorescent protein as a folding reporter

Protein folding can start right after the nascent chain is formed, but in case of membrane proteins, it is delayed as translation and membrane insertion are coupled and folding is completed after the last transmembrane segments have left the translocon (Luirink, von Heijne, Houben, & de Gier, 2005). In multidomain proteins, domains are tethered by either structured or disordered linkers and the rate of folding of individual domains can differ (Arviv & Levy, 2012; George & Heringa, 2002; Jappelli, Luzzago, Tataseo, Pernice, & Cesareni, 1992; Zhang & Ignatova, 2009). In the present method, green fluorescent protein (GFP) is used as a global folding reporter, which requires fusion of the fluorescent protein to the C-terminus of the target membrane protein. Thus, the synthesis of the membrane protein precedes that of the fluorescent protein. The folding of a membrane protein is typically faster than the folding and maturation of GFP, which takes about 30–90 min (Evdokimov et al., 2006; Reid & Flynn, 1997; Waldo, Standish, Berendzen, & Terwilliger, 1999). Therefore, when the target membrane protein is misfolded, it is likely to drag the not yet fully synthesized, folded, and/or matured GFP into an aggregated state. In contrast, when the target membrane protein is properly folded, the GFP β-barrel is formed and the chromophore will mature. Proper folding of the protein protects the chromophore from quenching by water dipoles, paramagnetic oxygen, or cis–trans isomerization (Tsien, 1998). The maturation process involves a series of covalent rearrangements of the amino acids that form the tripeptide chromophore (Ser/Thr65, Tyr66, and Gly67) within an α-helix that is buried inside the hollow β-barrel. When the chromophore matures, GFP becomes SDS resistant, and the protein migrates faster in SDS-PAA than the fully denatured polypeptide. The apparent difference in migration is about 10 kDa, and this difference is also observed when GFP is fused to another protein (Geertsma, Groeneveld, et al., 2008). Thus, when a membrane protein is well folded, the C-terminal GFP will fully mature, and the whole fusion protein migrates faster on SDS-PAA than the misfolded membrane protein–GFP fusion. By analyzing protein expression on immunoblots, one can easily discriminate the well-folded and misfolded proteins (Drew, Lerch, Kunji, Slotboom, & de Gier, 2006; Geertsma, Groeneveld, et al., 2008). Moreover, the fluorescence reports the absolute amount of folded fusion protein, which can be observed not only in gel but also in vivo (Drew et al., 2006; Linares et al., 2010).

We and others have observed that the in vivo activity of transport proteins correlates with the corresponding in-cell and in-gel GFP fluorescence of the fusion proteins (Geertsma, Groeneveld, et al., 2008; Hibi et al., 2008; Schlegel et al., 2012). In brief, the glutamate...

Produkt-Details

• Erscheint lt. Verlag: 6.4.2015
• Sprache: englisch
• Themenwelt: Medizin / Pharmazie
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
• ISBN-10: 0-12-801625-6 / 0128016256
• ISBN-13: 978-0-12-801625-1 / 9780128016251