Kinetic and Structural Differentiation of Trypsin from Different Origins

Abstract

Recombinant human insulin production involves expression of pre-pro insulin or insulin precursor in appropriate host system followed by extraction, conversion into insulin form, and further purification. The conversion from precursor forms into insulin is achieved using proteolytic enzyme trypsin or combination of trypsin and carboxypeptidase B. Because of trypsin’s specificity toward both arginine and lysine residues, this conversion often results in degradation products that can affect the final product quality.

The study presented herein was aimed at understanding the differences between porcine and bovine trypsin from both pancreatic and recombinant origins. The porcine trypsin from both pancreatic and recombinant sources was found to hydrolyse the substrates benzoyl-L-arginine ethyl ester hydrochloride (BAEE) or human insulin at a higher rate when compared to bovine trypsin. At a given pH and under identical conditions, recombinant porcine trypsin produced des-octapeptide insulin at a faster rate. Recombinant porcine trypsin was found to have the highest rate, the highest turnover number, and the lowest catalytic efficiency. There was significant amount of β sheets and random coil structures for all the trypsins studied with very low level of helical content observed in circular dichroism spectroscopy. The recombinant porcine trypsin had the highest helical content. Addition of divalent ions such as calcium and magnesium at 5 mM concentration decreased helical content.

Trypsin (EC 3.4.21.4) is a serine protease that exists widely in nature and has been discovered in bacteria, fungi, and mammals (1, 2). In mammals, trypsin is closely linked with metabolism, digestion, and coagulation. Trypsin is secreted from the pancreas as an inactive precursor (zymogen). Trypsin catalyses a hydrolytic cleavage of proteins and peptides at the carboxyl group of the basic amino acids arginine and lysine. It is generally used in leather processing, biotechnological processing, medicine, and food processing. Some of the prime applications in the biopharmaceutical industry include conversion of single chain pre-proinsulin or single-chain insulin precursor to two chains insulin, harvesting of cells by trypsinization in animal cell-culture works, and protein digestion in mass spectroscopy (3-5). Traditionally, trypsin has been mainly extracted from bovine and porcine pancreas. However, with the increase in demand for trypsin and problems due to its complex production process and potential contamination with infectious agents, heterologous expression has been carried out. Trypsin sequence from different sources has been expressed with different expression systems. Trypsin from varying origins such as bovine, porcine, and murine are commercially available both in natural and recombinant forms. With such a diversity of trypsin sources available, there is a need to understand the differences between these types of trypsin with respect to structure, specificity, and activity. Interestingly, it has been reported that trypsin-based protein digestion in mass spectroscopy depends on various factors such as origin, digestion conditions, and post-translational modifications of the protein (5). It has been reported that porcine trypsin has low electrophoretic mobility at pH 4.8 and high stability in alkaline media when compared to bovine and ovine trypsin based on their comparative studies on pancreatic trypsin of various origin (6, 7). Higher resistance of porcine trypsin to autocatalysis compared to bovine and ovine trypsin in presence of calcium ions has been reported (7). It was also deduced that porcine trypsin has lower isoelectric point (pI) than the other two trypsins (6).

The application of trypsin in insulin manufacturing industry is immense. The insulin and insulin analogs are typically expressed in precursor form, as pre-proinsulin in case of Escherichia coli (E. coli), or as single-chain precursor form in yeasts like Saccharomyces cerevisiae (S. cerevisiae) or Pichia pastoris (P. pastoris). Depending on the host organism, these forms are isolated/separated from cells and subjected to enzymatic conversion to insulin. Trypsin and carboxypeptidase B are widely used for this purpose. However, tryptic cleavage leads to the formation of many degradation products. Trypsin is an endoprotease (serine type) that cleaves peptide bonds at C-terminal end of arginine (Arg) or lysine (Lys) residues. Tryptic cleavage of pre-proinsulin molecules can occur at different cleavage sites simultaneously. Because of the many cleavage sides within a specific insulin precursor molecule, many undesired side-products can be formed during tryptic cleavage reaction (1) (see Figure 1).

Figure 1: Structural depiction of insulin and des-octa peptide insulin. (Note that amino acid residues B22-B30 are cleaved by trypsin). (Courtesy of the authors)

Based on the available information, it can be concluded that there are differences in the properties of trypsin from various sources including pI and electrophoretic mobility, among others. However, most of these studies are performed using the natural pancreatic trypsin, but there is little information available on how bovine and porcine trypsin from both pancreatic and recombinant origin may differ in their action. The present work focuses on generating sufficient evidence to understand these differences both with artificial substrate (BAEE) and recombinant human insulin as model substrates. The objective of this study is to understand how these trypsins are kinetically and structurally different, under a defined set of experimental conditions. The findings from this study may enable the insulin scientists immensely to control product-related impurities by choosing the appropriate trypsin. This control, coupled with techniques such as site directed mutagenesis, will help to increase the conversion efficiency of the insulin precursor form to insulin.
 

Materials and methods

Chemicals and enzymes
Recombinant porcine trypsin (expressed in P. pastoris) was purchased from Richcore Lifesciences Pvt Ltd. Recombinant bovine trypsin was purchased from BioGenomics (expressed in E. coli). Pancreatic porcine trypsin was purchased from Sigma Aldrich, and pancreatic bovine trypsin was purchased from Desert Biologicals. BAEE substrate and benzamidine HCl were purchased from Sigma Aldrich. Sodium phosphate monobasic monohydrate was purchased from J.T. Baker. Calcium chloride was purchased from Fisher Scientific, and acetonitrile (HPLC grade) was purchased from J.T Baker. 
 

Kinetic studies
Measurement of trypsin activity. The trypsin activity was measured as per the standard protocol (8). All optical density measurements were made using a microplate reader (BioTek).
 

The activity of trypsin was measured in BAEE units. One BAEE unit will produce a ΔA253 of 0.001 per min at pH 7.6 at 25 °C using BAEE as substrate (8).

Determination of V_max and K_m values. The apparent Michaelis-Menten constant (K_m), maximum velocity (V_max), and catalytic constant (K_cat) were determined. The BAEE concentration was varied from 0.029 to 0.7 mM. The kinetic parameters were then determined using a Lineweaver-Burk double-reciprocal plot. K_cat was calculated by the following equation: K_cat = V_max/ [E], where [E] is the enzyme concentration (M).

Secondary structure estimation using circular dichroism (far UV CD)Sample preparation for CD. The samples were prepared considering the key points given by Greenfield (9). The concentration of trypsin was maintained at 0.2 mg/mL. The trypsin samples were prepared in 5 mM phosphate buffer (pH 2.5). The spectrum was recorded using a spectrophotometer (JASCO Corporation, Tokyo Japan) at 25 °C in the wave length range between 190 and 250 nm with a constant nitrogen flush, using quartz cells of 1-mm path length. Each spectrum was analyzed with 5 mM phosphate buffer as blank. The spectrum for the secondary structure was analyzed using the Yang’s reference (10). Additionally, these trypsin samples were also incubated with 5 mM of CaCl2 and MgCl2 for 12 hours at 5±3 °C and analysed to understand the impact of these ions on structural changes. All the information was recorded at low pH because trypsin has a tendency to undergo autocatalysis at higher pH. 

Degradation of insulin by trypsin. The insulin API was extracted from the soluble drug product procured from market by dialyzing against 25 mM Tris HCl buffer of pH 9.0 using Spectra/Por 7 Pre-treated dialysis tubing (MWCO: 1 kD) (Spectrum Laboratories India). The dialyzed insulin concentration was measured using a high-performance liquid chromatography (HPLC) method developed based on earlier reports (11, 12). The method was operated with ACE C18-300 5-μ reverse-phase column (Advanced Chromatography Technologies) maintained at 40 °C. Mobile phase A consisted of 0.1% trifluoroacetic acid in ultrapure water and buffer B consists of 100% HPLC grade acetonitrile. A gradient elution was used as follows: 25% to 40% B in 4 column volumes (CVs), 40% to 60% B 0.5 CV, 60% B for 0.5 CV, 60% to 40%B in 0.75 CV, and 40% to 25% B in 1.25 CVs. A flow rate of 1.0 mL/min was maintained throughout. 

A modified procedure from Wang and Carpenter was used for trypsin digestion of recombinant human insulin (13). A fixed insulin concentration of 1mg/mL was considered for the study. This solution was maintained at pH of 9.0 in 50 mM Tris-HCl buffer system and 10 mM of CaCl2. To this solution, 250 BAEE units were added. Samples were taken every 30 minutes till 3 hours. Insulin standard was used for the calculation of the product content in the samples. 
 

Results and discussion

Kinetic studies using BAEE as substrate
All the experiments were performed in triplicates and the mean of the data was used to fit Lineweaver-Burk plot to calculate K_cat, K_m, and V_max values (Figure 2, Table I).

Figure 2: Line weaver-Burk plot for kinetic parameter determination. (Courtesy of the authors)

Table I. Kinetic parameters of trypsins using benzoyl–L–arginine ethyl ester hydrochloride (BAEE) as substrate values represents. V_max is maximum velocity. K_m Michaelis–Menten constant, K_cat is catalytic constant.

The V_max, K_m, K_cat, and K_cat/K_m values were found to vary from 4.15-52.55, 1.35-27.62, 3.53-10.95, and 39.65-548.30, respectively (Table I). Pancreatic porcine trypsin had the highest affinity (lowest Km value) and highest catalytic efficiency (K_cat/K_m = 218.39 x 105 1/mM.s), recombinant porcine trypsin appears to have the highest catalytic activity (Kcat = 10.95 x 105 s-1,) and lowest catalytic efficiency K_cat/K_m = 39.65 x 105 1/mM.s). However, it should be noted that use of Kcat/Km has its own pitfalls. Eisenthal et al. (14) have noted that an enzyme having a higher catalytic efficiency (i.e., K_cat/K_m value) can, at certain substrate concentrations, actually catalyse an identical reaction at lower rates than one having a lower catalytic efficiency. Both the bovine trypsin exhibit similar K_cat values. It should be noted that both the recombinant porcine and bovine trypsin were expressed in different hosts. Recombinant porcine trypsin expressed in Pichia system is extracellular whereas bovine trypsin expressed in E. coli aggregates as inclusion bodies. The kinetic values indicate that the host for expression and thereby different purification strategies do not affect the kinetic behavior and this solely depends on the sequence source, bovine or porcine. The V_max values indicate that both porcine trypsin have higher values compared to both bovine trypsin. In fact, recombinant porcine trypsin was almost eight times faster rate of hydrolysis when compared to pancreatic porcine trypsin. There is a clear distinction among porcine and bovine trypsin when V_max values are compared. K_m values observed are in line with the earlier observations made by Cunningham (15) (Km = 5 x10-2 mM value for the pancreatic bovine trypsin with BAEE as a substrate). 
 

Secondary structure elucidation using circular dichroism 
The secondary structure information recorded using far UV CD (Figure 3, Table II) indicates that trypsin from different sources predominately have random coil and β sheets. The helical content and turns accounted only for 10-15% in all these trypsin studied. Recombinant porcine trypsin had the highest helical content followed by pancreatic porcine trypsin. Interestingly, turns were absent in case of pancreatic bovine and porcine trypsin and present only in their recombinant sources. How these secondary structure differences influence the kinetic behavior needs to be understood further. An attempt made to record secondary structure information in presence of benzamidine hydrochloride as inhibitor did not result in reliable information. Jibson et al. (16) observed that trypsin spectra shows no evidence of conformational change accompanying the binding of either Bowman-Birk soybean inhibitor or chickpea inhibitor to the enzyme. These studies, however, were performed at neutral pH and in the presence of inhibitors. Further studies may be conducted with different inhibitors as noted by Jibson et al. (16) to understand the actual structural difference at alkaline pH. 

Figure 3: Far UV-CD data for all trypsins in the absence of metal ions. (Courtesy of the authors)
 

Sipos and Merkel (17) reported an increased structural stability of trypsin in presence of calcium ions. Secondary structure information was obtained for trypsin in presence of calcium and magnesium ions.
 

Table II. Secondary structure elements of all variants of trypsin in the absence and presence of ions (expressed as % values).

From Table II, it is evident that added calcium and magnesium reduced the helical content and increased sheets and random coils. It should be noted that all trypsins were pre-formulated at source with 20 mM CaCl₂. Presence of calcium salt possibly could have altered the structure already to some extent. Addition of calcium and magnesium most likely resulted in further decrease in the helical content. Calcium ions are known to reduce autocatalysis in trypsin. Sipos and Merkel (17) have proposed a possible conformational change to trypsin into a compact structure in presence of calcium ions. Thus, even though helix is somewhat disturbed when divalent ions are added, it may be that tertiary structure could still be compact to impart necessary protection against autolysis.
 

Degradation studies using insulinFigure 1 depicts the insulin and insulin des-octapeptide structure. Wang and Carpenter (13, 18) have noted that at pH 8 and 9, the arginyl bond in B chain is cleaved at a rate approximately 25 times that of lysyl bond. In this study pH of 9.0, it is expected that insulin des-octa peptide formation would occur. However, the difference in the rate of des-octa formation will be useful in understanding these trypsin activities further. The degradation data (Figure 4, Table III) indicates that recombinant porcine trypsin was able to form des-octapeptide at a faster rate than other trypsins. This observation indicate that recombinant porcine trypsin has higher affinity towards arginine residues than lysine residue. Similar observation was made during kinetic studies with BAEE where recombinant porcine trypsin showed the highest rate. Whereas, recombinant bovine trypsin had the slowest rate of insulin des-octapeptide formation among all the trypsins studied. This finding indicates that recombinant bovine trypsin has lower affinity toward arginine residue, and possibly higher affinity toward lysine residue. This observation reiterates some of the earlier observations on differences in various trypsins from different origins. Thus, applications that use trypsin need to become cognizant of such differences to maximize the throughput and reduce unwarranted side conversions. 

Figure 4: Rate of formation of des-octa peptide insulin by different trypsins. (Courtesy of the authors)
 

Table III. High-performance liquid chromatography (HPLC) purity values for insulin and insulin des-octapeptide during degradation study.

Conclusion

This study makes an effort to understand the kinetic and structural aspects of both porcine and bovine trypsin from pancreatic and recombinant sources. The authors have observed differences in turn over numbers among the four trypsins studied up to a magnitude of three-fold. The porcine trypsin from pancreas and recombinant source both showed higher affinity toward arginine substrates. The change in expression system for recombinant porcine or bovine trypsin did not seem to influence the kinetic behavior. The porcine trypsin, irrespective of source, behaved differently from bovine trypsin. The rates of cleavage of both BAEE and insulin were higher for porcine trypsin compared to bovine trypsin. The secondary structural information through far UV CD analysis indicates that trypsin, irrespective of its source, majorly displays random coils and β sheets with varied helical content. The helical content was the highest for recombinant porcine trypsin. It may be possible that these differences may be due to different purification processes used. The helical content of other recombinant bovine trypsin studied was lowest. The addition of divalent ions such as calcium and magnesium, even at low concentrations, seem to decrease helical content and increase random coil and β sheets. The implication of these changes needs to be confirmed at tertiary level because significant changes to structure at tertiary level can have significant impact on individual application. The study on degradation of insulin was able to underscore the differences in the rate of insulin des-octapeptide formation by different trypsins. The recombinant porcine trypsin had the highest rate. This observation is significant for applications such as insulin manufacturing because use of either porcine or bovine trypsin can lead to different levels of clippings at residues like B22 arginine and B29 lysine. Precise control of these impurities is essential for further purification stages. Thus, if the purification strategy requires greater control on des-octapeptide or impurity, then bovine trypsin should be preferred over porcine trypsin. It should be noted that of animal-derived enzymes in biologics manufacturing is highly controlled due to potential contamination.

Thus, recombinant bovine trypsins are always better option in this regard. Also, these findings may augment the differences observed in the proteomics profiles when using trypsin from different origins (5). 
 

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Article Details

BioPharm International
Vol. 30, No. 1
January 2017
Pages 38-43, 56

Citation

When referring to this article, please cite it as M. Aithal et al., "Kinetic and Structural Differentiation of Trypsin from Different Origins," BioPharm International 30 (1) January 2017.