Abstract
BackgroundOne of the major challenges for membrane protein structural genomics is establishing high-throughput cloning and expression screening methods to obtain enough purified protein in a homogeneous preparation for structural and functional studies. Here a series of ligation independent cloning based vectors were constructed to address this challenge.ResultsThe feasibility of these vectors was tested with 41 putative membrane proteins from Mycobacterium tuberculosis. The efficiency for direct cloning of these target genes from PCR products was 95% (39/41). Over 40% of cloned genes were overexpressed in Escherichia coli BL21 (DE3)-RP codon plus strain in the first round of expression screening. For those proteins which showed no expression, three protein fusion partners were prepared and it was found that each of the target proteins could be overexpressed by at least one of these fusions, resulting in the overexpression of two thirds of the cloned genes.ConclusionThis expression platform features high throughput cloning, high flexibility for different constructs, and high efficiency for membrane protein overexpression, and is expected to be useful in membrane protein structural and functional studies.
Highlights
One of the major challenges for membrane protein structural genomics is establishing high-throughput cloning and expression screening methods to obtain enough purified protein in a homogeneous preparation for structural and functional studies
There is no doubt that the construction of a high throughput cloning and expression screening platform would facilitate expression optimization through construction of fusion proteins and protein engineering as well as the characterization of membrane proteins. We have developed such a platform, which consists of a series of ligation independent clone (LIC) based vectors, for high throughput cloning and expression screening and tested the platform with 41 putative integral membrane proteins from Mycobacterium tuberculosis
These proteins range in molecular weight from 7.4 to 31 kDa with 1 to 4 putative transmembrane helices, similar to the target genes studied in a previous effort [2]
Summary
One of the major challenges for membrane protein structural genomics is establishing high-throughput cloning and expression screening methods to obtain enough purified protein in a homogeneous preparation for structural and functional studies. Genomic sequence analysis predicts that integral membrane proteins constitute 20–30% of all sequenced prokaryotic and eukaryotic genomes. These proteins are critical for many essential cellular functions and constitute 60 to 70% of current drug targets [1]. The remarkable gap between the significance of membrane proteins and the limited number of high resolution membrane protein structures is, in part, due to the availability of membrane proteins for structural biology. For structure determination, obtaining enough highly homogeneous protein is the first substantial challenge, especially for membrane proteins [3,4,5].
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