Abstract
The protein tyrosine phosphatase (PTPase) plays an important role in insect immune system. Our group has purified a type of acid phosphatase that could specifically dephosphorylate trans-Golgi p230 in vitro. In order to study this phosphatase further, we have identified and cloned the phosphatase gene from a locust specific Metarhizium anisopliae Strain CQMa102. The CQMa102 phosphatase was expressed in Pichia pastoris to verify its protease activity. The molecular weight (MW) and the isoelectric point (pI) of the phosphatase were about 85kDa and 6.15, respectively. Substrate specificity evaluation showed that the purified enzyme exhibited high activity on O-phospho-L-tyrosine. At its optimal pH of 6.5 and optimum temperature of 70°C, the protein showed the highest activity respectively. It can be activated by Ca2+, Mg2+, Mn2+, Ba2+, Co2+ and phosphate analogs, but inhibited by Zn2+, Cu2+, fluoride, dithiothreitol, β-mercaptoethanol and N-ethylmaleimide.
Highlights
The protein tyrosine phosphatase (PTPase) plays an important role in insect immune system
The amino acid sequence of the phosphatase shared identity with some alkaline phosphatases according to the NCBI database searching, the fact is that the purified phosphatase in our experiments had optimum activity at about pH 6.5 for
It is known that Zn2+ inhibition is an intrinsic property of specific phosphotyrosine protein phosphatases.24–27) The fact that Zn2+ strongly inhibited dephosphorylation activity in our experiments proved that the phosphatase displayed PTPase activity
Summary
The protein tyrosine phosphatase (PTPase) plays an important role in insect immune system. Our group has purified a type of acid phosphatase that could dephosphorylate trans-Golgi p230 in vitro. In order to study this phosphatase further, we have identified and cloned the phosphatase gene from a locust specific Metarhizium anisopliae Strain CQMa102. At its optimal pH of 6.5 and optimum temperature of 70 °C, the protein showed the highest activity respectively. Our group has purified an extracellular phosphatase from M. anisopliae It could dephosphorylate the signal transduction substance of locust humoral immunity in vitro20), which suggested that the fungus might interfere with the immune defense of locust. In order to clarify the characteristics of protein expression, the optimum pH of the purified phosphatase activity was studied and showed the phosphatase was an acid phosphatase. Substrate specificity and inhibitor sensitivity of the purified phosphatase displayed obvious protein tyrosine phosphatases activity. The phosphatase should be a novel acid phosphatase (AcP101) that displays protein tyrosine phosphatases activity
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