Construction of a series of vectors for high throughput cloning and expression screening of membrane proteins from Mycobacterium tuberculosis.
BACKGROUND
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. Here a series of ligation independent cloning based vectors were constructed to address this challenge.


RESULTS
The 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.


CONCLUSIONS
This 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.
Qin
Huajun
H
Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32310, USA. huajunq@yahoo.com
Hu
Jian
J
Hua
Yuanzhi
Y
Challa
Shridhar V
SV
Cross
Timothy A
TA
Gao
Fei P
FP
D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D011993:Recombinant Fusion Proteins