Seventy integral membrane proteins from the Mycobacterium tuberculosis genome have been cloned and expressed in Escherichia coli. A combination of T7 promoter-based vectors with hexa-His affinity tags and BL21 E. coli strains with additional tRNA genes to supplement sparsely used E. coli codons have been most successful. The expressed proteins have a wide range of molecular weights and number of transmembrane helices. Expression of these proteins has been observed in the membrane and insoluble fraction of E. coli cell lysates and, in some cases, in the soluble fraction. The highest expression levels in the membrane fraction were restricted to a narrow range of molecular weights and relatively few transmembrane helices. In contrast, overexpression in insoluble aggregates was distributed over a broad range of molecular weights and number of transmembrane helices.

Apolipoprotein E is a 299-residue lipid carrier protein produced in both the liver and the brain. The protein has three major isoforms denoted apoE2, apoE3, and apoE4 which differ at positions 112 and 158 and which occur at different frequencies in the human population. Genome-wide association studies indicate that the possession of two apoE4 alleles is a strong genetic risk factor for late-onset Alzheimer’s disease (LOAD). In an attempt to identify a small molecule stabilizer of apoE4 function that may have utility as a therapy for Alzheimer’s disease, we carried out an NMR-based fragment screen on the N-terminal domain of apoE4 and identified a benzyl amidine based fragment binder. In addition to NMR, binding was characterized using various other biophysical techniques, and a crystal structure of the bound core was obtained. Core elaboration ultimately yielded a compound that showed activity in an IL-6 and IL-8 cytokine release assay.

Sixteen of 22 low molecular weight integral membrane proteins from Mycobacterium tuberculosis with previously poor or undetectable levels of expression were expressed in Escherichia coli as fusions with both the maltose-binding protein (MBP) and a His 8 -tag. 68% of targeted proteins were expressed in high yield (≥30 mg/L) in soluble and/or inclusion body form. Thrombin cleavage of the MBP fusion protein was successful for 10 of 13 proteins expressed as soluble proteins and for three proteins expressed only as inclusion bodies. The use of autoinduction growth media increased yields over Luria-Bertani (LB) growth media in 75% of the expressed proteins. Expressing integral membrane proteins with yields suitable for structural studies from a set of previously low and nonexpressing proteins proved highly successful upon attachment of the maltose-binding protein as a fusion tag.Production of membrane proteins for structural and functional studies remains a difficult task due to their highly hydrophobic nature [1,2]. During the last decade increasing efforts to express prokaryotic and eukaryotic membrane proteins have resulted in significant advancements for the expression of several bacterial transporter proteins [3][4][5][6][7][8][9], outer membrane proteins of bacteria [10], membrane protein complexes [11][12][13] and a few eukaryotic G-protein coupled receptors [14][15][16][17][18][19][20][21]. With others we have successfully expressed more than 70 membrane proteins from Mycobacterium tuberculosis in Escherichia coli using a His-tag [22]. However, a significant number of tested proteins (28%) were not expressed at detectable (Coomassie stain or Western) levels in E. coli. Expression of nonmembrane proteins by the Methanobacterium thermoautotrophicum structural genomics pilot project showed that almost Corresponding author: T.A. Cross. National High Magnetic Field Laboratory. 1800 E. Paul Dirac Dr. Tallahassee, Fl. 32310. Email: Email: cross@magnet.fsu.edu. Phone: 850-644-0917. Fax: 850-644-8350. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 50% of cloned genes did not express when an N-terminal His-tag was used [23]. More importantly, 41% of our cloned membrane proteins with a His-tag that have a molecular weight less than 13.7 kDa did not express, and only 22% expressed well, such that a Coomassie stain was observed representing enough protein to proceed with structural studies. Here, we have set out to enhance the expression of small molecular weight proteins and to identify a robust protocol for doing this. NIH Public AccessWhile many different vectors and tag...

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