title: Studies on the expression and processing of human proinsulin derivatives encoded by different DNA constructs.
abstract: A synthetic gene encoding human proinsulin, containing Escherichia coli preferred codons, with an additional N-terminal methionine, was used for the expression, of M-proinsulin and construction of nine derivatives. No improvement in expression was noted, relative to that of M-proinsulin, when the 5'- of the gene was appended to codons for seven amino acids of a well expressed E. coli protein (threonine dehydrogenase), or the constructs contained multiple copies of the proinsulin gene. That in the latter constructs only the gene adjacent to the prometer sequence is expressed, was shown by a construct containing a proinsulin gene followed by that for interferon α-2b. With the latter construct, the proinsulin was, predominantly, expressed. The availability of data on the constructs prompted, subjecting these to analysis by two models designed to predict the expression of proteins from the sequences, of putative mRNA, around the start of translation but no significant relationship was noted. In all cases the proteins were expressed as inclusion bodies, which were refolded to give products of desired masses and successfully converted into insulin derivatives. Of all the constructs containing a trypsin sensitive site before phenylalanine (F), the N-terminal sequence, MKR↓F, was most efficiently processed, by a cocktail of trypsin and buffalo carboxypeptidase B, to give insulin with the removal of the N-terminus linker as well as the C-peptide in a single step, without cleaving the trypsin sensitive K(29)T(30) peptide bond.
authors.affiliation: School of Biological Sciences, University of the Punjab, New Campus, Lahore 54590, Pakistan.
authors.forename: Farheen
authors.initials: F
authors.lastname: Aslam
authors.forename: Qurra-tul Ann Afza
authors.initials: QT
authors.lastname: Gardner
authors.forename: Hina
authors.initials: H
authors.lastname: Zain
authors.forename: Muhammad Shahid
authors.initials: MS
authors.lastname: Nadeem
authors.forename: Muhammad
authors.initials: M
authors.lastname: Ali
authors.forename: Naeem
authors.initials: N
authors.lastname: Rashid
authors.forename: Muhammad
authors.initials: M
authors.lastname: Akhtar
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002020:Buffaloes; D043424:Carboxypeptidase B / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D058849:Protein Refolding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011384:Proinsulin; D000429:Alcohol Oxidoreductases; C023159:L-threonine 3-dehydrogenase; D043424:Carboxypeptidase B; D014357:Trypsin
keywords: Bioassay of insulin/proinsulin; Conversion of proinsulin into insulin; Free energy of mRNA unfolding/interaction and protein expression; M-proinsulin/M-insulin; Mass spectra of proinsulin/insulin; Protein refolding; native proteins containing N-terminal methionine; showing cleavage between the peptide bond, single-letter code is generally used for the amino acid residues; ↓