Effect of the codon following the ATG start site on the expression of ovine growth hormone in Escherichia coli.
For expression of ovine growth hormone (OGH) in inclusion bodies without an affinity histidine tag at either end of the protein, three clones, differing only in the second codon following the ATG start site, were constructed. Their expression was studied by SDS-PAGE followed by immunoblotting. Clone Ala.OGH (clone 1), beginning with Met.Ala.Phe.Pro ellipsis, did not show any expression. Clone Phe.OGH (clone 3), beginning with Met.Phe.Pro ellipsis, gave very high levels of OGH expression following IPTG induction. However, in clone Gly.OGH (clone 2), in which the Ala codon was replaced with a Gly codon at the second position after the start site, a lower level of expression was obtained. Northern hybridization analysis showed that upon IPTG induction, OGH mRNA was transcribed from all three clones. These results therefore, imply that lack of expression in clone 1 and a lower level of expression in clone 2 are not due to a failure of transcription; however, they may be due to inefficient initiation of translation. The secondary structure analysis of mRNA predicts inaccessibility of different elements of the RBS in the case of Ala.OGH (clone 1). The present study highly underscores the importance of mRNA secondary structure at the start site in regulation of expression of a cloned gene in Escherichia coli, a prokaryotic expression system.
Puri
N
N
Embryo Biotechnology Laboratory, Product Development Cell, Gene Regulation Laboratory, National Institute of Immunology, JNU Complex, Aruna Asaf Ali Marg, New Delhi, 110 067, India.
Appa Rao
K B
KB
Menon
S
S
Panda
A K
AK
Tiwari
G
G
Garg
L C
LC
Totey
S M
SM
D000818:Animals; D003062:Codon / Q000235:genetics / Q000494:pharmacology*; D018387:Codon, Initiator / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000235:genetics; D005822:Genetic Vectors / Q000737:chemistry; D013006:Growth Hormone / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis / Q000187:drug effects; D012756:Sheep / Q000235:genetics* / Q000502:physiology; D014158:Transcription, Genetic / Q000187:drug effects; D014169:Transformation, Bacterial
D003062:Codon; D018387:Codon, Initiator; D012333:RNA, Messenger; D013006:Growth Hormone