Development of mutant human immunoreactive trypsinogen 1 (IRT1) and mutant human immunoreactive trypsinogen 2 (IRT2) for use in immunoassays
Introduction
Immunoreactive Trypsinogen (IRT) is the proenzyme form of the serine protease trypsin that is secreted from human pancreas for protein digestion [ [1]]. It has three major isoforms, IRT1 (UniProt P07477, GenBank NG_008307), IRT2 (UniProt P07478, GenBank NG_008322) and IRT3 (UniProt P35030, GenBank NG_001337). In this study, IRT1 and IRT2 are studied due to their usage as diagnostic markers for CF [1,2]. The main difference between IRT1 and IRT2 is their ionic charge. IRT1 is cationic while IRT2 is anionic [3]. IRT is not in the active form as first secreted from pancreas [4,5]. After it is transported to the small intestine, it is activated and converted to trypsin via enterokinases, proteolytic cleavage, pH alteration, and auto-activation [6]. The inactive form is called as trypsinogen, and the active type is called as trypsin [7]. IRT is normally found in the bloodstream, but in some cases, such as cystic fibrosis, the level in the blood can rise. Significant changes in IRT concentration in the bloodstream can be used as a diagnostic marker in Cystic Fibrosis (CF) diagnosis for newborn screening [8]. Because there is no significant difference between IRT levels of adult CF patients and healthy people, so IRT is not an option for adult diagnosis. However, IRT detection tools for newborn diagnosis are useful options [1]. In addition to CF, recent articles point out to the potential of IRT usage as a diagnostic marker in pancreatitis and several cancer types such as ovarian, gastric and colorectal cancers [7,[9], [10], [11]]. It is essential to develop a sensitive, cost-effective and easy method for efficient monitoring and early diagnosis of these diseases. ELISA tests are commonly used in the screening programs, and the efficiencies of diagnostic tests are key factors of reliable diagnosis of the diseases. For the detection of Cystic fibrosis, protein-based ELISA kits are widely used [1]. In every ELISA test, a calibration curve, which is determined by using the standard protein, is needed to be constructed to measure the concentration of the unknown sample [12]. However, usage of active trypsin limits the efficiency of the test since it cleaves the antibodies used as the recognition material [[13], [14], [15]].
ELISA tests depend on antigen-antibody interaction, thus the reactive components used in the analysis are mostly proteins [16]. In the developed kits, IRT is used for the establishment of the standard curve for quantification. IRT samples used in ELISA are measured in a buffer that is extracted heel-prick blood absorbed membrane [6,17].
In this study, it was aimed to deactivate human IRT by mutating the amino acid residue at the cleavage site of trypsinogen into active trypsin and hereby block its protease activity to make easier its use in immunoassay studies. Because using active trypsin limits the efficiency of the test since it cleaves the antibodies used as recognition material. IRT has an active side at the 23rd position which can lead to silencing the activity if mutated [18]. To prevent the protease activity of IRT, the lysine residue at the 23rd position was changed to aspartic acid. The aim of the study is different from previous studies because it is the first IRT activity silencing in order to use in immunoassays for both two isoforms via lysine-aspartic acid alteration. It is significant to use two different antigenic targets because in diagnostic immunoassay kits expanding diagnostic targets can minimize false negative or false positive results. Usage of IRT1 and IRT2 in CF detection kits are already commonly-used options [7,9].
Consistently with the aim, the results that show mutant proteins don't have catalytic activity were proven via enzymatic activity results. Enzymatic activities of the proteins were measured with Nα-BenzoylL-arginine ethyl ester hydrochloride (BAEE) which is a trypsin substrate. It was found that the enzymatic activity of IRT is diminished. In parallel, the antigenic activity of two IRT isoforms was assessed by ELISA and Western Blot techniques.
Section snippets
Materials and methods
Design of Sequence: Both IRT1 and IRT2 have activation sites (Fig. 1) at the same position and mutating their activation peptides is the main aim to block its activity. To produce K23D IRT1 and K23D IRT2, coding DNA sequences (CDS) were designed, synthesized and cloned in multiple cloning sites of the pGAPZα A yeast expression vector. The products were produced as being fused with 6xHis Tag at C terminus. The yeast expression plasmids containing alpha factor secretion signal, IRT CDS and 6xHis
Production of IRT1 & IRT2
In this study, newly designed IRT1 and IRT2 genes were successfully cloned, confirmed by sequencing and transformed to P. pastoris X-33 strain. Survived colonies were grown in liquid culture and evaluated by using ELISA in terms of IRT producing capabilities (Fig. 3(A), Fig. 3(B)).
For IRT1, two of the 23 colonies were selected which gave the highest signal in the ELISA, and a subculture was performed every 24 h for 96 h to determine the optimum culture times of the IRT1 proteins that were
Discussion
Detection and quantification of protease enzymes in protein-based assays is a challenge since the analyte may cleave assay components [[19], [20], [21]]. The proteolytic activity of the proteases can be inhibited by the use of protease inhibitors or deactivation of the active side. In this study, blocking the autoactivation ability with K23D mutation was selected for the purpose of avoiding proteolytic activity. The study describes a method to produce a recombinant human immune reactive
Conclusions
In this study, recombinant human IRTs have K23D mutation, which changes lysine (K) residue at the 23rd position to aspartic acid (D). This mutation inhibits IRT activation and proteolytic activity by preventing the cleavage of inactive trypsinogen to active trypsin. The antigenic properties of mutant proteins were tested and confirmed by ELISA (Enzyme-Linked Immunosorbent Assay) and Western Blot. It has been shown that mutant IRTs have similar antigenic properties as wild-types, except the
Funding
This study was supported by TÜBİTAK 1003-SBAG, project number 115S124.
CRediT authorship contribution statement
Mustafa Divyapicigil: Investigation, Writing - original draft. Serife Seyda Pirincci Gokturk: Conceptualization, Methodology, Writing - review & editing. Bengu Ergenoglu: Investigation. Fatima Yucel: Project administration. Esin Aslankaraoglu Akcael: Supervision.
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