Hyperexpression of rat spermatidal protein TP2 in Escherichia coli by codon optimization and engineering the vector-encoded 5' UTR.
We have recently reported the cDNA cloning of rat spermatidal protein TP2 and its expression in Escherichia coli using pTrc 99A as the expression vector. However, the expression level was very low. We have now improved the expression of TP2 over fivefold by (1) optimizing the codons for lysine, arginine, proline, leucine, glycine, valine, threonine, alanine, and tyrosine and (2) by engineering the vector-encoded 5' UTR. The expressed protein was in the soluble phase and could be purified to homogeneity by successive chromatography on Zinc-NTA-agarose affinity matrix and heparin agarose. Serendipitously, we have also observed a concomitant hyperinduction of vector encoded beta-lactamase gene along with TP2 in the E. coli BL21 (DE3) cells.
Department of Biochemistry, Indian Institute of Science, Bangalore-, 560 012, India.
A R
AR
Meetei
M R
MR
Rao
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002868:Chromosomal Proteins, Non-Histone / Q000096:biosynthesis* / Q000235:genetics; D003062:Codon; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D008297:Male; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D015202:Protein Engineering / Q000379:methods*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012270:Ribosomes / Q000378:metabolism; D013087:Spermatids / Q000737:chemistry*
D002868:Chromosomal Proteins, Non-Histone; D003062:Codon; D011994:Recombinant Proteins; C053886:spermatid transition proteins