Expression enhancement of bubaline somatotropin in E. coli through gene modifications in the 5'-end coding region.
This study addresses the problem of poor expression of somatotropin (ST) gene in E. coli and describes expression enhancement through silent and non-silent gene modifications. A series of constructs with codon optimization, substitution, deletion or addition in the 5'-region of the sequence encoding bubaline ST (BbST) were prepared. In the native form, the BbST expression was barely discernible on SDS-gel of the total E. coli cellular proteins (TCP). Introduction of silent and non-silent mutations in +2 to +8 codons, however, raised the expression levels to varying extents. In some constructs, a single base variation, i.e., G-->A or G-->C led to a remarkable increase in BbST expression (up to 28% of the TCP) whereas in the case of G-->T substitution the expression dropped to undetectable levels. Deletion of native GCC codon and addition of CAUCAC repeat thrice at +2 position enhanced the expression up to 48%, while insertion of NGG codons at the same position caused just a modest increase in expression. Differences in expression appeared as if related to the nature of early downstream codons (especially +2) and the stability of mRNA secondary structure although the levels of intracellular mRNA pools, as analyzed by real-time RT-PCR were quite similar. Overall, the study highlights the importance of 5'-end codon adaptations in solving the problems encountered in expressing the eukaryotic genes in E. coli.
Sadaf
Saima
S
School of Biological Sciences, University of the Punjab, Lahore, Pakistan.
Khan
Muhammad Altaf
MA
Akhtar
Muhammad Waheed
MW
D000818:Animals; D001483:Base Sequence; D002020:Buffaloes / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics*; D013006:Growth Hormone / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; D011994:Recombinant Proteins; D013006:Growth Hormone