scholarly journals Construction of a full length α-factor secretory signal sequence for human insulin precursor expression in Pichia pastoris

2021 ◽  
Vol 762 (1) ◽  
pp. 012066
Author(s):  
N Utami ◽  
D Nurdiani ◽  
Hariyatun ◽  
E W Putro ◽  
W Kusharyoto
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Signal Sequence ◽  
Full Length ◽  
Insulin Precursor

scholarly journals Secretory expression of human insulin precursor in Pichia pastoris employing truncated α-factor leader sequence and a short C-peptide

2018 ◽  
Vol 23 (2) ◽  
pp. 102 ◽  
Author(s):  
Dini Nurdiani ◽  
Hariyatun Hariyatun ◽  
Wien Kusharyoto
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Lifestyle Changes ◽  
Diabetic Patients ◽  
Scale Production ◽  
Middle Income ◽  
C Peptide ◽  
Insulin Precursor

In the past ten years, diabetes prevalence has increased rapidly in low- and middle-income countries due to lifestyle changes. This increased number of diabetic patients leads to the escalation of recombinant insulin demand, which is creating a large global insulin market. Pichia pastoris has appeared as an alternative host to produce recombinant proteins. It has excellent qualifications as an expression host for large-scale production of recombinant proteins for therapeutic use. In this study, we attempted to express the insulin precursor (IP) in P. pastoris. We used a synthetic IP-encoding gene constructed in frame with the truncated α-factor secretory signal and a short C-peptide (DGK) linked A- and B-chain of human insulin in a pD902 expression vector. Several zeocin resistant clones were successfully obtained and verified with PCR using AOX1 specific primers for the integration of the expression cassette into the P. pastoris genome and for the identification of Mut phenotypes. The secretion of IP by the Pichia pastoris clone in the culture supernatant was confirmed using SDS-PAGE, where a single band of the secreted IP with a molecular mass above 6.5 kDa was found.

scholarly journals Transformation and characterization of human insulin precursor gene in Pichiapastoris X-33

2021 ◽  
Vol 948 (1) ◽  
pp. 012084
Author(s):  
F C Sekaringtyas ◽  
D Hardianto ◽  
N Karimah ◽  
V Nida ◽  
A Zahra
Keyword(s):  
Escherichia Coli ◽  
Pichia Pastoris ◽  
Human Insulin ◽  
Agar Medium ◽  
Transformation Process ◽  
Efficient Production ◽  
Purification Process ◽  
Insulin Production ◽  
Insulin Precursor

Abstract The case of diabetes increases significantly and has been projected to reach 592 million people in 2035. Consequently, the necessity of insulin will rise manifold and an efficient production system for insulin production is required to meet the market demands. The human insulin precursors that enzymatically converted to human insulin can be produced using Escherichia coli, Saccharomyces cerevisiae, or Pichia pastoris. In this study, Pichia pastoris is used for production human insulin precursor because the resulting recombinant protein can be folded accordingly and secreted to the external environment of the cell that simplifies the purification process. The study was initiated with the insertion of a synthetic gene of human insulin precursor into the pPICZaA to create recombinant pPICZaA-IP plasmid. The recombinant plasmid was transformed into Escherichia coli Top10 which then isolated and digested by the SacI enzyme. The linearize pPICZaA-IP plasmid was transfected into Pichia pastoris X-33 by electroporator. The result of transformation process, a total of 20 colonies of P pastoris X-33 were selected and inoculated in YPD agar medium containing Zeocin. The two colonies of P pastoris were characterized by PCR and sequencing showed that the recombinant pPICZaA-IP plasmid was successfully integrated into selected colonies of P pastoris.

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Rafid A. Abdulkareem
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Expression Vector ◽  
Expression System ◽  
Insulin Gene ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Major Band ◽  
Human Insulin Gene ◽  
Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

From secretion in Pichia pastoris to application in apple juice processing: Exo-polygalacturonase from Sporothrix schenckii 1099-18

2021 ◽  
Vol 28 ◽  
Author(s):  
Ersin Karataş ◽  
Ahmet Tülek ◽  
Mehmet Mervan Çakar ◽  
Faruk Tamtürk ◽  
Fatih Aktaş ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Apple Juice ◽  
Biochemical Characterization ◽  
Signal Sequence ◽  
Sporothrix Schenckii ◽  
Juice Clarification ◽  
Pectinolytic Enzyme ◽  
Pectinolytic Enzymes ◽  
Protein Expression And Purification ◽  
Wide Range

Background: Polygalacturonases are a group of enzymes under pectinolytic enzymes related to enzymes that hydrolyse pectic substances. Polygalacturonases have been used in various industrial applications such as fruit juice clarification, retting of plant fibers, wastewater treatment drinks fermentation, and oil extraction. Objectives: The study was evaluated at the heterologous expression, purification, biochemical characterization, computational modeling, and performance in apple juice clarification of a new exo-polygalacturonase from Sporothrix schenckii 1099-18 (SsExo-PG) in Pichia pastoris. Methods: Recombinant DNA technology was used in this study. Two different pPIC9K plasmids were constructed with native signal sequence-ssexo-pg and alpha signal sequence-ssexo-pg separately. Protein expression and purification performed after plasmids transformed into the Pichia pastoris. Biochemical and structural analyses were performed by using pure SsExo-PG. Results: The purification of SsExo-PG was achieved using a Ni-NTA chromatography system. The enzyme was found to have a molecular mass of approximately 52 kDa. SsExo-PG presented as stable at a wide range of temperature and pH values, and to be more storage stable than other commercial pectinolytic enzyme mixtures. Structural analysis revealed that the catalytic residues of SsExo-PG are somewhat similar to other Exo-PGs. The KM and kcat values for the degradation of polygalacturonic acid (PGA) by the purified enzyme were found to be 0.5868 µM and 179 s-1, respectively. Cu2+ was found to enhance SsExo-PG activity while Ag2+ and Fe2+ almost completely inhibited enzyme activity. The enzyme reduced turbidity up to 80% thus enhanced the clarification of apple juice. SsExo-PG showed promising performance when compared with other commercial pectinolytic enzyme mixtures. Conclusion: The clarification potential of SsExo-PG was revealed by comparing it with commercial pectinolytic enzymes. The following parameters of the process of apple juice clarification processes showed that SsExo-PG is highly stable and has a novel performance.

scholarly journals Expression of novel acidic lipase from Micrococcus luteus in Pichia pastoris and its application in transesterification

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Selfela Restu Adina ◽  
Antonius Suwanto ◽  
Anja Meryandini ◽  
Esti Puspitasari
Keyword(s):  
Fatty Acid ◽  
Pichia Pastoris ◽  
Specific Activity ◽  
Signal Sequence ◽  
Micrococcus Luteus ◽  
Acid Methyl Ester ◽  
Industrial Applications ◽  
Expression Level ◽  
Lipase Gene ◽  
Acidic Lipase

Abstract Background Lipases are promising biocatalysts for industrial applications and attract attention to be explored. A novel acidic lipase has been isolated from the lipolytic bacteria Micrococcus luteus EMP48-D (LipEMP48-D) screened from tempeh. The lipase gene had previously been overexpressed in Escherichia coli BL21, but the expression level obtained was relatively low. Here, to improve the expression level, the lipase gene was cloned to Pichia pastoris. We eliminated the native signal sequence of M. luteus and replaced it with α-mating factor (α-MF) signal sequence. We also optimized and synthesized the lipase gene based on codon preference in P. pastoris. Results LipEMP48-D lipase was expressed as an extracellular protein. Codon optimization has been conducted for 20 codons, with the codon adaption index reaching 0.995. The highest extracellular lipase activity obtained reached 145.4 ± 4.8 U/mg under AOX1 promoter in P. pastoris KM71 strain, which was 9.7-fold higher than the previous activity in E. coli. LipEMP48-D showed the highest specific activity at pH 5.0 and stable within the pH range 3.0–5.0 at 40 °C. LipEMP48-D also has the capability of hydrolyzing various long-chain triglycerides, particularly olive oil (100%) followed by sunflower oil (88.5%). LipEMP48-D exhibited high tolerance for various polar organic solvents with low log P, such as isopropanol (115.7%) and butanol (114.6%). The metal ions (Na+, K+, Ca2+, Mg2+, Mn+) decreased enzyme activity up to 43.1%, while Fe2+ increased relative activity of enzymes up to 200%. The conversion of free fatty acid (FFA) into fatty acid methyl ester (FAME) was low around 2.95%. Conclusions This study was the first to report overexpression of Micrococcus lipase in yeast. The extracellular expression of this acidic lipase could be potential for biocatalyst in industrial fields, especially organic synthesis, food industry, and production of biodiesel.

Sign in / Sign up

Export Citation Format

Share Document