4272

4043

كارشناسي ارشد

بيوتكنولوژي كشاورزي

اصفهان : دانشگاه صنعتي اصفهان، دانشكده كشاورزي

1387

پانزده، 139، [II] ص. : مصور، جدول

ص. ع. به: فارسي و انگليسي

رضا زارعي

23/7/1387

بدر الدين ابراهيم سيد طباطبائي، مسعود بهار

مهندسي كشاورزي

ID4043

به فارسي و انگليسي: قابل رويت در نسخه ديجيتال

Abstract In resent years peptides have been shown to be more important that what it waspreviously believed in both plant and animal growth and development This as well asthe pharmaceutical and industrial applications of peptides have led to improved largescale production and purification of these compounds Generally production of peptidesintended for in vitro research regulation of cell physiology and therapeutic applications follows one of two main methods chemical synthesis or hetrologous expression Yetexpression of very short polypeptide chains can sometimes be problematic in microbialsystems including in bacterial cells such as Escherichia coli In these cases the use oftandem repeat of peptide units or insertion of fusion partners may increase expressionefficiency In the present study MBP8298 a 17 amino acid peptide was selected as amodel peptide for a study of heterologous expression in E coli Nucleotide codingsequence of MBP8298 was designed and synthesized based on the bacterial codon usage mRNA secondary structure as well as the required nucleotide restriction sites andpolypeptide cleavage points A nucleotide sequence encoding a fusion partner based onthe sequence by Paulus et al 2004 and hence named Paulus Sequence was alsoconstructed the same gene synthesis technology The fusion partner was subsequentlyattached upstream of two concatemers of MBP8298 coding sequence that in turn had adownstream 6His tag coding sequence This construct dubbed PM2H comprising Paulus 2 MBP8298 6His was then cloned into a pET 21c expression vector The PaulusSequence encodes an AU rich sequence just after the start codon followed by a stem loop in the mRNA structure and is reported to increase expression of downstreampolypeptides probably because of enhanced translation initiation For comparisonpurposes two other constructs were also prepared First one named M2H that had asimilar structure to PM2H but lacked the Paulus Sequence and second another onenamed UM2H that contained ubiquitin ORF Isolated from Medicago truncatula inplace of the Paulus Sequence Once sequencing of the constructs were confirmed theywere transformed into the expression hosts BL21 DE3 and subsequently induced withIPTG to produce the recombinant proteins Analysis of expressed proteins by SDS PAGE indicated that UM2H and PM2H were present as soluble and inclusion bodyrespectively No expression of M2H was detected Expressed fusion proteins werepurified by Ni affinity batch chromatography which in turn verified the expectedpresence of C terminal His tags In summary it was shown that both fusion partners Paulus Sequence and ubiqutin markedly increase the MBP8298 expression level thatwas otherwise non detectable This report constitutes the first description of recombinantexpression of MBP8298

رضا زارعي

بدر الدين ابراهيم سيد طباطبائي، مسعود بهار