Effects of second-codon mutations on expression of the insulin-like growth factor-II-encoding gene in Escherichia coli

Cantrell, Amanda; Burgett, Stanley G.; Cook, James A.; Smith, Michèle C.; Hsiung, Hansen M.

doi:10.1016/0378-1119(91)90176-c

Cited by 8 publications

(5 citation statements)

References 20 publications

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“…The resulting plasmid expressed abundant CPT activity (results not shown), indicating that the lack of response with the human constructs (Figure 2) stemmed from a peculiarity of the human CPT II sequence itself. It is known that codon usage can radically alter the efficiency of expression of foreign proteins in E. coli [16,17] and that the problem with poor expression can often be overcome by the use of a fusion protein construct [18]. Accordingly, the sequence for mature human CPT II was placed downstream of a sequence encoding 27 kDa of a GST (plasmid pGST/HMet25).…”

Section: Resultsmentioning

confidence: 99%

Expression of precursor and mature carnitine palmitoyltransferase II in Escherichia coli and in vitro: differential behaviour of rat and human isoforms

Brown

Sen

Soltis³

et al. 1993

Biochemical Journal

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cDNAs corresponding to the precursor and mature forms of rat carnitine palmitoyltransferase II (CPT II) were found to be readily expressed in Escherichia coli. In both cases, catalytically active immunoreactive protein was produced and became largely membrane-associated. The precursor form of the enzyme was not proteolytically processed. Removal of 126 bp from the 5' end of the cDNA coding region allowed expression of a truncated CPT II (lacking the N-terminal 17 residues of the mature protein), but this product was inactive. cDNAs encoding the precursor and mature forms of human CPT II resisted direct expression in E. coli. However, the impediment was overcome when the latter cDNA was ligated in-frame 3' to sequence encoding a glutathione S-transferase. This construct yielded abundant quantities of the corresponding fusion protein, a portion of which was soluble and catalytically active. In vitro transcription and translation of the various cDNAs established that the lower mobility on SDS/PAGE of rat CPT II compared with its human counterpart (despite their identical numbers of amino acids) is an intrinsic property of the primary sequences of the proteins themselves. Also, the human cDNA was found to contain an artifactual termination signal for T3 RNA polymerase that could be bypassed by the T7 polymerase. Thus rat CPT II can be expressed in active form in E. coli with characteristics similar to those of the enzyme in mitochondria, opening the way to future location of active sites within the molecule. An alternative expression system will be needed for similar studies on human CPT II.

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Section: Resultsmentioning

confidence: 99%

Expression of precursor and mature carnitine palmitoyltransferase II in Escherichia coli and in vitro: differential behaviour of rat and human isoforms

Brown

Sen

Soltis³

et al. 1993

Biochemical Journal

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“…Analogue 1 contains a cysteine bridge switch (9-46, 47-51) in two of the three native cysteine bridges (originally 9-47, 21-60 and 46-51), and was prepared as described (Smith et al, 1989). Analogue 2, which contains the N-terminal addition Met-His-Trp, was overexpressed in Escherichia coli and purified as described (Cantrell et al, 1991).…”

Section: Materials and Methods Materialsmentioning

confidence: 99%

Altered affinity of insulin-like growth factor II (IGF-II) for receptors and IGF-binding proteins, resulting from limited modifications of the IGF-II molecule

Beukers

Pham

et al. 1991

Biochemical Journal

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The binding affinities of seven analogues of recombinant human insulin-like growth factor II (hIGF-II) were characterized for the IGF type-I and type-II receptors and insulin receptors, as well as for IGF-binding protein (IGFBP)-1, IGFBP-2, IGFPB-3 and human serum IGFBPs. A switch of two of the three cysteine bridges in hIGF-II, 9-47 and 46-51 to 9-46 and 47-51, severely impaired the binding of this analogue to all receptors and to the IGFBPs. The affinities for the IGF type-I receptor and the IGFBPs were decreased over 100-fold, while the binding to the insulin receptor and the IGF type-II receptor was less affected, with a 6-10-fold decrease in affinity. Slight modifications of the N-terminus had only minor effects upon the binding of hIGF-II to the IGFBPs or to the receptors. Deletion of both the N-terminal amino acid and the two C-terminal amino acids resulted in moderate decreases in affinity, with a 60% decrease in affinity for IGFBP-1 and the IGF type-I receptor. Acetylation of the N-terminus of Ala1 and the epsilon-nitrogen of Lys65 decreased the affinity, by 60-90%, of hIGF-II for all of the IGFBPs and receptors. The experiments involving acetylation of IGF-II or switching of its cysteine bridges indicated that these modifications (no substitution, deletion or addition of any of the 67 amino acids of hIGF-II) may lead to a severe impairment of the binding affinity of IGF-II for both the IGFBPs and the receptors. Acetylation of the epsilon-nitrogen of Lys65, which causes a charge change, or alteration of the three-dimensional structure, as shown by the cysteine bridge switch, lead to a severe impairment of the binding affinity for the binding proteins and for the receptors. In general, care should be taken with the synthesis of analogues and the interpretation of resulting binding data, since affinity alterations ascribed to amino acid changes may instead be caused by alterations of the charge or the three-dimensional structure of the protein.

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“…This is consistent with the results reported for studies of Met-X,,-Trp-IGF I1 mutants in E. coli. (Cantrell et al, 1991).…”

Section: Table 2 Expression Levels Of Hpi and Their Analogsmentioning

confidence: 99%

“…The data was inconclusive with regard to the expression levels. Therefore, we studied the mRNA stabilities of Met-X,,-IGF-I derivatives by Northern Blot analysis as described by Cantrell et al (1991). The data (not shown) suggested that the mRNA stability of high producers were greater than that of low producers.…”

Section: Table 2 Expression Levels Of Hpi and Their Analogsmentioning

confidence: 99%

“…coli cells containing recombinant plasmids were grown at 30 "C to early logarithmic phase and shifted to 42 "C as described above. Total cellular RNA was isolated as described previously (Cantrell et al, 1991), separated by electrophoresis on a 1% agarose/ formaldehyde gel and blotted onto nitrocellulose paper. The RNA was hybridized with 32p-labeled IGF-1 or hPI gene and quantitated by densitometry of the autoradiograms.…”

Section: Rna Isolation and Blottingmentioning

confidence: 99%

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Increased production of low molecular weight recombinant proteins in Escherichia coli

Belagaje

Reams

et al. 1997

Protein Science

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A general method for obtaining high-level production of low molecular weight proteins in Escherichia coli is described. This method is based on the use of a novel Met-X,,-protein construction which is formed by insertion of a single amino acid residue (preferably Arginine or Lysine) between the N-terminal methionine and the protein of interest. The utility of this method is illustrated by examples for achieving high-level production of human insulin-like growth factor-1, human proinsulin, and their analogs. Furthermore, highly produced insulin-like growth factor-] derivatives and human proinsulin analogs are converted to their natural sequences by removal of dipeptides with cathepsin C.

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Effects of second-codon mutations on expression of the insulin-like growth factor-II-encoding gene in Escherichia coli

Cited by 8 publications

References 20 publications

Expression of precursor and mature carnitine palmitoyltransferase II in Escherichia coli and in vitro: differential behaviour of rat and human isoforms

Expression of precursor and mature carnitine palmitoyltransferase II in Escherichia coli and in vitro: differential behaviour of rat and human isoforms

Altered affinity of insulin-like growth factor II (IGF-II) for receptors and IGF-binding proteins, resulting from limited modifications of the IGF-II molecule

Increased production of low molecular weight recombinant proteins in Escherichia coli

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