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Query: UNIPROT:P05231 (interleukin-6)
 23,907 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have designed an expression vector for the secretion of human interleukin-6 (hIL-6) in which the mature protein is fused through a spacer peptide, containing a KEX-2 like protein processing signal, to the entire Aspergillus niger glucoamylase (glaA) gene. Transformation of Aspergillus nidulans with this vector results in fungal strains secreting equimolar amounts of the glucoamylase and IL-6 proteins. The KEX2-type processing signal, Lys-Arg, is recognized and cleaved efficiently by an enzyme present in A. nidulans resulting in the secretion of an authentic mature hIL-6 protein at levels of up to 5 mg/l.
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PMID:Efficient KEX2-like processing of a glucoamylase-interleukin-6 fusion protein by Aspergillus nidulans and secretion of mature interleukin-6. 136 12

To develop improved methods for heterologous protein production in Aspergillus niger, we studied the secretion of human interleukin-6 (hIL6). Since in vitro experiments with culture medium revealed that hIL6 was rapidly degraded, several protease-deficient strains of A. niger were isolated and tested for reduced degradation of hIL6 compared with the wild-type strain. The mutant strain giving the least degradative effect on hIL6 (designated AB1.13) was transformed with several hIL6-expression plasmids. Initially, hIL6 was expressed using various signal sequences fused to the sequence of mature hIL6. The resulting transformants did not produce detectable amounts of hIL6, despite high transcription levels in one transformant. We hypothesized that hIL6 was not efficiently processed during passage along the secretion pathway. Therefore, hIL6 was expressed as a fusion protein with glucoamylase, a protein which is efficiently secreted by A. niger and expression of which can easily be measured enzymatically. To obtain mature hIL6, a sequence encoding the KEX2 cleavage-site (Lys-Arg) was inserted between glucoamylase and hIL6 sequences. Mature active hIL6 was found to be secreted in the extracellular medium. Using this combined approach of transforming a protease-deficient strain with a fusion construct containing the KEX2 site, up to 15 mg l-1 active hIL6 was obtained in shake-flask culture. A fusion construct without the KEX2 site resulted in substantially higher production of the fusion protein, but hIL6 was not active in the fused form. These results indicate that A. niger contains a protease with similar specificity as the KEX2 protease from yeast.
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PMID:Secretion of heterologous proteins by Aspergillus niger: production of active human interleukin-6 in a protease-deficient mutant by KEX2-like processing of a glucoamylase-hIL6 fusion protein. 776 98

In this study we have analyzed the effects of a glucoamylase gene fusion on the mRNA levels and protein levels for the human interleukin-6 gene (hil6) and the guar alpha-galactosidase gene (aglA). Previously it was shown that production of nonfused alpha-galactosidase and hIL-6 in Aspergillus awamori was limited at transcriptional and (post)translational levels, respectively (R. J. Gouka, P. J. Punt, J. G. M. Hessing, and C. A. M. J. J. van den Hondel, Appl. Environ. Microbiol. 62:1951-1957, 1996). Vectors were constructed which contained either the hil6 or aglA gene fused to the Aspergillus niger glucoamylase gene (glaA) under control of the efficient 1,4-beta-endoxylanase A promoter and transcription terminator. For comparison, the vectors were integrated in a single copy at the pyrG locus of A. awamori. A glaA fusion to the 5' end of the hil6 gene resulted in a large increase in hIL-6 yield, whereas with a glaA fusion to the 3' end of the hil6 gene, almost no protein was produced. Nevertheless, the steady-state mRNA levels of both fusions were very similar and not clearly increased compared to those of a strain expressing nonfused hIL-6. Fusions of glaA to the 5' end of the wild-type guar aglA gene resulted in truncated mRNA lacking almost 900 bases (> 80%) of the aglA sequence. When the coding sequence of the wild-type aglA gene was replaced by a synthetic aglA gene with optimized Saccharomyces cerevisiae codon usage, full-length mRNA was obtained. Compared to a nonfused synthetic aglA gene, a glaA fusion with the synthetic aglA gene resulted in a 25-fold increase in the mRNA level and, as a consequence, a similar increase in the alpha-galactosidase protein level. The truncated transcripts derived from the wild-type aglA gene were further analyzed by nuclear run-on transcription assays. These experiments indicated that transcription elongation in the nucleus proceeded at least 400 bases downstream of the site where the truncation was determined, indicating that transcription elongation or premature termination was not the reason for the generation of truncated mRNAs. As the truncated mRNA also contained a poly(A) tail, truncation most likely occurs by incorrect processing of the aglA mRNA in the nucleus.
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PMID:Glucoamylase gene fusions alleviate limitations for protein production in Aspergillus awamori at the transcriptional and (post) translational levels. 902 27

Secreted yields of foreign proteins may be enhanced in filamentous fungi through the use of translational fusions in which the target protein is fused to an endogenous secreted carrier protein. The fused proteins are usually separated in vivo by cleavage of an engineered Kex2 endoprotease recognition site at the fusion junction. We have cloned the kexin-encoding gene of Aspergillus niger (kexB). We constructed strains that either overexpressed KexB or lacked a functional kexB gene. Kexin-specific activity doubled in membrane-protein fractions of the strain overexpressing KexB. In contrast, no kexin-specific activity was detected in the similar protein fractions of the kexB disruptant. Expression in this loss-of-function strain of a glucoamylase human interleukin-6 fusion protein with an engineered Kex2 dibasic cleavage site at the fusion junction resulted in secretion of unprocessed fusion protein. The results show that KexB is the endoproteolytic proprotein processing enzyme responsible for the processing of (engineered) dibasic cleavage sites in target proteins that are transported through the secretion pathway of A. niger.
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PMID:Characterization of the kexin-like maturase of Aspergillus niger. 1061 49