Multiple joined genes prevent product degradation in Escherichia coli.

Abstract

A method is described that allows the expression of a stable human proinsulin product in Escherichia coli as encoded by either a fused or an unfused gene construction. In the fused system, the human proinsulin coding sequence is joined to the 3' side of a fragment containing the lac promoter and the coding sequence for a small part of the NH2 terminus of fi-galactosidase. In the unfused system, the proinsulin coding sequence is linked directly to a fragment containing the Tac promoter followed by a bacteria! Shine-Dalgarno sequence. In both systems, the human proinsulin product is too unstable to be detected by NaDodSO4/polyacrylamide gel electrophoresis or even pulse-chase analysis. However, when multiple copies of the proinsulin coding sequence are tandemly linked such that the resultant protein product contains multiple copies of the proinsulin domain, the stability of the product is markedly increased in both the fused and the unfused expression systems. In the unfused system, three tandemly linked proinsulin polypeptide domains are required for stabilization, whereas two proinsulin domains plus the bacterial leader protein enhance stability in the fused system. The polypeptide product of a multiple copy proinsulin gene can be cleaved into single proinsulin units by cyanogen bromide treatment.Recombinant DNA technology has been used extensively for the development of bacterial strains expressing useful eukaryotic products such as human insulin (1, 2), human growth hormone (3), interferon (4j, and viral vaccines (5-7).

Increased expression in Escherichia coli of a synthetic gene encoding human somatomedin C after gene duplication and fusion

“…Recently, similar self-fusion experiments with the human proinsulin gene have been reported (22). Although the folded structures of SMC and proinsulin are related (1), we believe that our results confirm the generality of this approach to the stabilization of gene products of low molecular weight in E.…”

Bacitracin significantly reduces degradation of peptides in plant cell cultures

“…There are several strategies for the recovery of these proteins in an undegraded state. The protein can in some cases be protected from degradation if it is produced as a polyprotein (Shen, 1984), or as a hybrid with a more stable protein.…”

Bioproduction and self-assembly of designer peptides

“…One effective approach used to increase the proportion of target peptide in the expressed product is the incorporation of multiple copies of the target sequence. [310][311][312][313] However, the number of incorporated repeats cannot be increased indefinitely, and the higher repeat number constructs tend to display lower expression. [310][311][312][313] The optimal repeat numbers for expression must therefore be determined empirically for each expression system.…”

“…312,314 To further enhance potential bioproduction yields, peptides may be expressed as tandem repeats or "concatemers" in the absence of a fusion partner. 313 In this approach the target peptide comprises almost the entire expressed product. Similar to multiple copies in fusion proteins, this approach also requires empirical optimisation of repeat numbers.…”

“…Similar to multiple copies in fusion proteins, this approach also requires empirical optimisation of repeat numbers. [313][314][315] Furthermore, expression of such designed constructs will generally succeed only if the resulting concatemer can form a stably folded structure. Targeted design approaches such as those outlined in Section 1.2 may be utilised to give peptide and concatemer sequences that will fold into stable structures for expression.…”