Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastorisan alpha 1-antitrypsin in Pichia pastoris

Abstract

Background: Human alpha 1-antitrypsin (AAT) is a potent inhibitor of multiple serine proteases, and protects tissues against their harmful effects. Individuals with reduced or abnormal production of this inhibitor need intravenous administration of exogenous protein. In this study, we employed the methylotrophic (methanol utilizing) yeast Pichia pastoris (P. pastoris) as a preferential host for efficient production and secretion of recombinant AAT. Furthermore, we examined different strategies to maximize the yield of the secreted protein.Results: Our findings revealed that optimizing the codon usage of AAT gene for P. pastoris had positive effects on the level of secreted AAT under the control of inducible alcohol oxidase 1 (AOX1) and constitutive glycerol aldehyde phosphate dehydrogenase (GAP) promoters. Compared to AOX1, the GAP promoter increased the yield of AAT by more than two fold. It was also demonstrated that the human AAT native signal sequence was more effective than the well-known yeast signal sequence, alpha mating factor (α-MF). Doubling gene dosage nearly doubled the production of AAT, though dosages exceeding this limit had negative effects on the yield. Conclusion: P. pastoris is shown to be an efficient expression system for production of recombinant and biologically active AAT. Also different strategies could be used to elevate the amount of this secretable protein.

Identification and characterization of N-glycosylation site on a Mucor circinelloides aspartic protease expressed in Pichia pastoris: effect on secretion, activity and thermo-stability

“…As an illustration, we compared these sequences including the first 10 amino acids just after the cleavage site to 54 native signal peptide sequences of P. pastoris, which were recently predicted (De Schutter et al 2009 ), and some residues of the α-MF pro-sequence (Waters et al 1988 ; He et al 2012 ) matched with N-terminal amino acids of a mature protein of P. pastoris . In addition, we observed similar results with the glucoamylase (Chen et al 2007 ), pectate lyase B (Guo et al 1995 ), laccase (Liu et al 2003 ), alpha-1-antitrypsin (Arjmand et al 2013 ), urine protein complex (Ferrari et al 1997 ), acetylcholinesterase (Morel and Massoulie 1997 ) and alpha amylase signal peptide (Li et al 2011 ).…”

Enhancement of Alpha 1-antitrypsin Production in Pichia pastoris by Designing and Optimizing Medium Using Elemental Analysis

“…The activity of the produced recombinant AAT in the diff erent media was determined by the elastase inhibition assay according to the previously described method (22) with slight modifi cations. N-Succinyl-Ala (3)-ρ-nitroanilide (S4760, Sigma) was used as an elastase substrate.…”

Co‐expression of alpha‐1 antitrypsin with cytoplasmic domain of v‐SNARE in Pichia pastoris: Preserving biological activity of alpha‐1 antitrypsin

“…The E. coli strain DH5α was used for bacterial transformation and recombinant plasmids propagation. Low salt broth (LB) was used in recombinant bacteria culture (0.5% w/v yeast extract, 1% w/v tryptone, 0.5% w/v NaCl with Zeocin 25 μg/mL) . Pichia pastoris cells were grown overnight in YPD broth (2% peptone, 1% yeast extract, 2% dextrose) at 30 C°/250 rpm.…”

“…The purity and molecular weight of proteins were assessed by SDS‐PAGE on a 12% polyacrylamide gel . To detect recombinant A1AT and soluble domain of v‐SNARE, western blot was applied . First, the proteins were transferred to a Hybond‐P polyvinylidene difluoride (PVDF) membrane.…”

“…Yet, the number of studies that has examined the expression of A1AT in methylotrophic yeasts is quite limited. Some studies have reported secretory expression of A1AT in P. pastoris ; however, they are not comprehensive and further investigation is required to examine the credibility of results . In contrast to the most research papers that have examined extracellular expression to simplify the consequent downstream processes, in this paper, the intracellular expression was considered as a more appropriate choice.…”