Abstract
BackgroundDifficult to express peptides are usually produced by co-expression with fusion partners. In this case, a significant mass part of the recombinant product falls on the subsequently removed fusion partner. On the other hand, multimerization of peptides is known to improve its proteolytic stability in E. coli due to the inclusion of body formation, which is sequence specific. Thereby, the peptide itself may serve as a fusion partner and one may produce more than one mole of the desired product per mole of fusion protein. This paper proposes a method for multimeric production of a human alpha-fetoprotein fragment with optimized multimer design and processing. This fragment may further find its application in the cytotoxic drug delivery field or as an inhibitor of endogenous alpha-fetoprotein. ResultsMultimerization of the extended alpha-fetoprotein receptor-binding peptide improved its stability in E. coli, and pentamer was found to be the largest stable with the highest expression level. As high as 10 aspartate-proline bonds used to separate peptide repeats were easily hydrolyzed in optimized formic acid-based conditions with 100% multimer conversion. The major product was represented by unaltered functional alpha-fetoprotein fragment while most side-products were its formyl-Pro, formyl-Tyr, and formyl-Lys derivatives. Single-step semi-preparative RP-HPLC was enough to separate unaltered peptide from the hydrolysis mixture. ConclusionsA recombinant peptide derived from human alpha-fetoprotein can be produced via multimerization with subsequent formic acid hydrolysis and RP-HPLC purification. The reported procedure is characterized by the lower reagent cost in comparison with enzymatic hydrolysis of peptide fusions and solid-phase synthesis. This method may be adopted for different peptide expression, especially with low amino and hydroxy side chain content.
Highlights
Difficult to express peptides are usually produced by co-expression with fusion partners
The number of inserts was analyzed by Polymerase chain reaction (PCR) with T7 promotor and terminal primers followed by agarose electrophoresis
Pentameric peptide expression improves yield Recombinant expression of short polypeptides tends to be complicated mostly due to the low proteolytic stability in E. coli
Summary
Difficult to express peptides are usually produced by co-expression with fusion partners. This paper proposes a method for multimeric production of a human alpha-fetoprotein fragment with optimized multimer design and processing. Enzymatic digestion with factor Xa (IEGR↓), enterokinase (DDDDK↓), thrombin (LVPR↓GS), TEV protease (ENLYFQ↓G), and HRV 3C protease (LEVLFQ↓GP) is the most common methods for hydrolysis of fusion proteins [6,7,8,9]. These methods are highly specific, but limited to enzyme costs and sensitivity to environmental changes. Denaturing conditions can be detrimental to enzyme activity, while most
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