Abstract：
:The expression of recombinant proteins in bacterial hosts may alter the biophysical properties of the protein of interest as a result of differences in post-translational processing from that observed when produced in the native cell. For example, recombinant human interleukin-1beta (IL-1beta) is produced as three isoforms when expressed in the Escherichia coli strain BL-21(DE3). These isoforms are produced by the non-homogeneous processing of the N-terminal methionine residue by the endogenous bacterial aminopeptidase and differ in the first residue (1-met, 1-ala, and 1-pro). Importantly, these isoforms have significantly different binding affinities for the IL receptor protein. Varying the temperature, media composition, and point of induction affects this N-terminal processing to favor one of the three isoforms of IL-1beta. We found changes in media composition and/or point of induction affected the abundance of the isoforms by as much as 15-fold. The 1-pro isoform decreased from 60.9 to 4.7% in Luria broth (LB) and minimal media (MM), respectively, when protein expression was induced at an OD600 of 0.9. Conversely, the abundance of the 1-met isoform is much higher in MM than in LB showing the reverse effect (2.6 and 50.7% in LB and MM, respectively, at an OD600 of 0.9), and the degree to which they are favored depends significantly upon the induction point. Our results show that it is possible to favor the expression of various N-terminal isoforms of IL-1beta by adjusting the environmental growth conditions. Given that the initiator methionine residue is necessary for expression in bacterial hosts and is known to affect the stability of other recombinant proteins our approach may be a useful general method for determining the optimal conditions for expressing and purifying pure, homogenous samples of recombinant proteins for structural and biological studies.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Covalt JC Jr,Cao TB,Magdaroag JR,Gross LA,Jennings PAdoi
10.1016/j.pep.2005.01.010keywords:
subject
Has Abstractpub_date
2005-05-01 00:00:00pages
45-52issue
1eissn
1046-5928issn
1096-0279pii
S1046-5928(05)00021-5journal_volume
41pub_type
杂志文章abstract：:Leptin is a 16-kDa nonglycosylated hormone that is produced in mature adipocytes and which acts primarily in the hypothalamus to reduce food intake and body weight. While the rat is a representative laboratory animal model in obesity research, so far recombinant rat leptin was not available. In the present study, rat ...
journal_title：Protein expression and purification
pub_type： 杂志文章
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
pub_type： 杂志文章
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
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journal_title：Protein expression and purification
pub_type： 杂志文章
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journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2003.11.005
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journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2000.1290
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