Dataset Information

Expression and antimicrobial activity of the recombinant bovine lactoferricin in Pichia pastoris.

ABSTRACT: Lactoferricin, a multifunctional peptide located in the N-terminal region of lactoferrin, has a broad-spectrum bacteriostatic activity. It is a promising candidate as a food additive and immune fortification agent and does not have the risks associated with drug residues and drug resistance. First, we performed promoter and host cell screening to achieve the recombinant expression of lactoferricin in Pichia pastoris, showing an initial titer of 19.5 mg/L in P. pastoris X-33 using P_AOX1 promoter. Second, we constructed a 0030-α hybrid signal peptide by fusing the 0030 signal peptide with the pro-sequence of α-factor secretory signal peptide. This further increased the production of lactoferricin, with a titer of 28.8 mg/L in the fermentation supernatant in the shaking flask. Next, we increased the expression of lactoferricin by fusing it with anionic antioxidant peptides. The neutralization of positive charges yielded a titer of 55.3 mg/L in the shaking flask, and a highest titer of 193.9 mg/L in a 3-L bioreactor. The antimicrobial activity analysis showed that recombinant-expressed lactoferricin exhibited potent antibacterial activity against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. This study provides a reference for the construction of microbial cell factories capable of efficiently synthesizing antimicrobial peptides.

SUBMITTER: Lv X

PROVIDER: S-EPMC10787256 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Expression and antimicrobial activity of the recombinant bovine lactoferricin in Pichia pastoris.

Lv Xueqin X Zhang Yuting Y Wang Lingrui L Cui Shixiu S Liu Yanfeng Y Li Jianghua J Du Guocheng G Liu Long L

Synthetic and systems biotechnology 20231225 1

Lactoferricin, a multifunctional peptide located in the N-terminal region of lactoferrin, has a broad-spectrum bacteriostatic activity. It is a promising candidate as a food additive and immune fortification agent and does not have the risks associated with drug residues and drug resistance. First, we performed promoter and host cell screening to achieve the recombinant expression of lactoferricin in Pichia pastoris, showing an initial titer of 19.5 mg/L in P. pastoris X-33 ...[more]

PMID: 38221910

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Project description:BackgroundWhen performing functional and structural studies, large quantities of pure protein are desired. Most membrane proteins are however not abundantly expressed in their native tissues, which in general rules out purification from natural sources. Heterologous expression, especially of eukaryotic membrane proteins, has also proven to be challenging. The development of expression systems in insect cells and yeasts has resulted in an increase in successful overexpression of eukaryotic proteins. High yields of membrane protein from such hosts are however not guaranteed and several, to a large extent unexplored, factors may influence recombinant expression levels. In this report we have used four isoforms of aquaporins to systematically investigate parameters that may affect protein yield when overexpressing membrane proteins in the yeast Pichia pastoris.ResultsBy comparing clones carrying a single gene copy, we show a remarkable variation in recombinant protein expression between isoforms and that the poor expression observed for one of the isoforms could only in part be explained by reduced transcript levels. Furthermore, we show that heterologous expression levels of all four aquaporin isoforms strongly respond to an increase in recombinant gene dosage, independent of the amount of protein expressed from a single gene copy. We also demonstrate that the increased expression does not appear to compromise the protein folding and the membrane localisation.ConclusionsWe report a convenient and robust method based on qPCR to determine recombinant gene dosage. The method is generic for all constructs based on the pPICZ vectors and offers an inexpensive, quick and reliable means of characterising recombinant P. pastoris clones. By using this method we show that: (1) heterologous expression of all aquaporins investigated respond strongly to an increase in recombinant gene dosage (2) expression from a single recombinant gene copy varies in an isoform dependent manner (3) the poor expression observed for AtSIP1;1 is mainly caused by posttranscriptional limitations. The protein folding and membrane localisation seems to be unaffected by increased expression levels. Thus a screen for elevated gene dosage can routinely be performed for identification of P. pastoris clones with high expression levels of aquaporins and other classes of membrane proteins.

Project description:UnlabelledΒACKGROUND: The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains.ResultsA fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production.ConclusionsCo-overexpressing enzymes of the methanol utilization pathway significantly affected the specific growth rate, the methanol uptake and the specific productivity of recombinant P. pastoris MutS strains. A recently developed methodology to determine strain specific parameters based on dynamic batch cultivations proved to be a valuable tool for fast strain characterization and thus early process development.

Dataset Information

Expression and antimicrobial activity of the recombinant bovine lactoferricin in Pichia pastoris.

Publications

Expression and antimicrobial activity of the recombinant bovine lactoferricin in <i>Pichia pastoris</i>.

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