The functional and biological significance of translation initiation context sequence in determin... more The functional and biological significance of translation initiation context sequence in determining high-level expression of modified synthetic human α1-proteinase inhibitor (α1-PI) gene was documented in stable transgenic tomato plants. Context sequence of initiator ATG codon derived from statistical analysis of databases was identified as taaA(A/C)aATGGCt in highly expressed dicot plant genes. Removal of initiator ATG context sequence reduced the expression of recombinant α1-PI protein to fourfolds. The mutation of consensus base at +4 position to a pyrimidine either alone or with substitution at −3 position eliminated most of the α1-PI expression, while mutation at −3 alone resulted in about sevenfold reduction. The presence of steady-state levels of α1-PI transcript in transgenic plants indicated that the variation in expression is entirely due to the point mutations incorporated in translation initiation context. These results indicated the significance of conserved nucleotide sequence around initiator ATG codon in augmenting post-transcriptional events and high-level expression of heterologous genes in transgenic plants.
Human ␣ 1 -proteinase inhibitor (␣ 1 -PI) is the most abundant protease inhibitor found in the bl... more Human ␣ 1 -proteinase inhibitor (␣ 1 -PI) is the most abundant protease inhibitor found in the blood and expression of biologically active recombinant ␣ 1 -PI has great potential in therapeutic applications. We report here the expression of a synthetic ␣ 1 -PI gene and its variants in Escherichia coli. Modified ␣ 1 -PI gene and its single amino acid variants were cloned in pMAL-c2X vector, which allowed expression of recombinant protein(s) as a fusion of maltose-binding protein (MBP) with factor Xa protease recognition site between the fusion partners. The synthetic gene(s) were expressed in different E. coli strains and maximum expression of recombinant ␣ 1 -PI and variants up to 24% of total soluble protein (TSP) was achieved with engineered strain carrying extra copies of tRNAs for rare codons. Recombinant ␣ 1 -PI protein(s) were purified by amylose affinity chromatography with high homogeneity and overall yield of about 7-9 mg l −1 of bacterial culture (∼5.2 g wet cell mass). E. coli expressed recombinant ␣ 1 -PI showed specific anti-elastase activity and appeared as a single band of ∼45.0 kDa on SDS-PAGE. Primary structure of purified protein and integrity of N-terminus has been verified by mass spectrometric analysis. Recombinant ␣ 1 -PI expressed in E. coli was fully intact having molecular mass similar to native unglycosylated protein purified from human plasma. Increased thermostability and specific activities of purified ␣ 1 -PI variant proteins confirmed the stabilizing effect of incorporated mutations. Our results demonstrate efficient expression and purification of stable and biologically active ␣ 1 -PI and its variants in E. coli for further therapeutic applications.
The functional and biological significance of translation initiation context sequence in determin... more The functional and biological significance of translation initiation context sequence in determining high-level expression of modified synthetic human α1-proteinase inhibitor (α1-PI) gene was documented in stable transgenic tomato plants. Context sequence of initiator ATG codon derived from statistical analysis of databases was identified as taaA(A/C)aATGGCt in highly expressed dicot plant genes. Removal of initiator ATG context sequence reduced the expression of recombinant α1-PI protein to fourfolds. The mutation of consensus base at +4 position to a pyrimidine either alone or with substitution at −3 position eliminated most of the α1-PI expression, while mutation at −3 alone resulted in about sevenfold reduction. The presence of steady-state levels of α1-PI transcript in transgenic plants indicated that the variation in expression is entirely due to the point mutations incorporated in translation initiation context. These results indicated the significance of conserved nucleotide sequence around initiator ATG codon in augmenting post-transcriptional events and high-level expression of heterologous genes in transgenic plants.
Human ␣ 1 -proteinase inhibitor (␣ 1 -PI) is the most abundant protease inhibitor found in the bl... more Human ␣ 1 -proteinase inhibitor (␣ 1 -PI) is the most abundant protease inhibitor found in the blood and expression of biologically active recombinant ␣ 1 -PI has great potential in therapeutic applications. We report here the expression of a synthetic ␣ 1 -PI gene and its variants in Escherichia coli. Modified ␣ 1 -PI gene and its single amino acid variants were cloned in pMAL-c2X vector, which allowed expression of recombinant protein(s) as a fusion of maltose-binding protein (MBP) with factor Xa protease recognition site between the fusion partners. The synthetic gene(s) were expressed in different E. coli strains and maximum expression of recombinant ␣ 1 -PI and variants up to 24% of total soluble protein (TSP) was achieved with engineered strain carrying extra copies of tRNAs for rare codons. Recombinant ␣ 1 -PI protein(s) were purified by amylose affinity chromatography with high homogeneity and overall yield of about 7-9 mg l −1 of bacterial culture (∼5.2 g wet cell mass). E. coli expressed recombinant ␣ 1 -PI showed specific anti-elastase activity and appeared as a single band of ∼45.0 kDa on SDS-PAGE. Primary structure of purified protein and integrity of N-terminus has been verified by mass spectrometric analysis. Recombinant ␣ 1 -PI expressed in E. coli was fully intact having molecular mass similar to native unglycosylated protein purified from human plasma. Increased thermostability and specific activities of purified ␣ 1 -PI variant proteins confirmed the stabilizing effect of incorporated mutations. Our results demonstrate efficient expression and purification of stable and biologically active ␣ 1 -PI and its variants in E. coli for further therapeutic applications.
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Papers by Shweta Jha