High‐throughput screening of soluble recombinant proteins

Abstract

The aims of high-throughput (HTP) protein production systems are to obtain well-expressed and highly soluble proteins, which are preferred candidates for use in structure-function studies. Here, we describe the development of an efficient and inexpensive method for parallel cloning, induction, and cell lysis to produce multiple fusion proteins in Escherichia coli using a 96-well format. Molecular cloning procedures, used in this HTP system, require no restriction digestion of the PCR products. All target genes can be directionally cloned into eight different fusion protein expression vectors using two universal restriction sites and with high efficiency (>95%). To screen for well-expressed soluble fusion protein, total cell lysates of bacteria culture (∼1.5 mL) were subjected to high-speed centrifugation in a 96-tube format and analyzed by multiwell denaturing SDS-PAGE. Our results thus far show that 80% of the genes screened show high levels of expression of soluble products in at least one of the eight fusion protein constructs. The method is well suited for automation and is applicable for the production of large numbers of proteins for genome-wide analysis. . Cloning and expression in Escherichia coli are favored in many instances because E. coli has relatively simple genetics, is well characterized, has a relatively rapid growth rate, and has few post-translational protein modifications. One disadvantage, however, of expressing heterologous proteins in E. coli is that proteins are frequently expressed as insoluble aggregated folding intermediates, known as inclusion bodies (Paul et al. 1983). Although it may be possible to increase protein solubility by optimizing expression condition or by refolding the recombinant proteins, in the interests of throughput, only a single set of growth or folding conditions can be used.Gene fusion is another approach that has been successfully used for producing soluble heterologous proteins in E. coli (Uhl'en and Moks 1990). Several carrier proteins are widely used in gene fusion, including thioredoxin (Trx), maltose-binding protein (MBP), glutathione S-transferase (GST), intein, calmodulin-binding protein (CBP), NusA, and cellulose-associated protein (CAP). Although the use of these carrier proteins has resulted in the successful overexpression of many heterologous proteins, each was tested Reprint requests to: Ting-Fang Wang, Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, Republic of China; e-mail: tfwang@ gate.sinica.edu.tw; fax: 886-2-27889759. 1These two authors contributed equally to this work.

Self-Assembly of MinE on the Membrane Underlies Formation of the MinE Ring to Sustain Function of the Escherichia coli Min System

“…It is a small protein that can be dissected into three functional regions: the N-terminal membrane-targeting motif (MinE [2][3][4][5][6][7][8][9][10][11][12] ), the MinD-interacting domain (MinE [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31], and the C-terminal dimerization domain (MinE ). Recent evidence suggests that MinE exhibits dynamic conformational flexibility upon association with different interacting partners.…”

Optimisation of expression and purification of the feline and primate foamy virus transmembrane envelope proteins using a 96 deep well screen

“…A strong influence of the fusion partner on protein expression and solubility has been described repeatedly [36][37][38]. In order to facilitate testing for solubility of the produced protein, an additional centrifugation step using filter plates or an ultracentrifugation step using 96 well plates may also be included to remove insoluble material [38][39][40].…”

Production of recombinant cellulase enzyme from Pleurotus ostreatus (Jacq.) P. Kumm. (type NRRL-0366)

“…PCR reaction was performed in a total volume of 50 l and contain 5 l (5 x Green Go Taq flexi buffer (promega, USA ), 5 l (5 x colorless Go Taq flexi buffer (Promega, USA), (100 mM Tris-HCl (pH 8.8 at 25°C) 500 mM KCl),5 l MgCl2 (25 Mm) ( promega, USA), 2 l 4 dNTPs mixture (10 mM of each) (BIORON, Germany), 4 l C. DNA of chymosin,4 l of each primer (20 pmol / l), 2 U Taq polymerase (5 U / l) (promega, USA), the reaction volume was completed to 50 l with sterile distilled H2O. The reaction mixtures were subjected to Daba et al 1199 amplification as follows: initial denaturation step at 95°C for 3 min, followed by 35 cycles of amplification with denaturation at 95°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min ending with extension at 72°C for 10 min, the thermocycler (PTC-200 Peltier, USA) was used for performing PCR amplification, as described by Shih et al (2002).…”