Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris

Shi, Xinjie; Karkut, Tammy; Chamankhah, Mahmood; Alting-Mees, Michelle A.; Hemmingsen, Sean M.; Hegedus, Dwayne D.

doi:10.1016/s1046-5928(02)00706-4

Cited by 135 publications

(108 citation statements)

References 36 publications

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“…Shi et al [98] identified three types of proteases present in P. pastoris culture expressing a single-chain antibody (scFv) targeted against Mamestra configurata serpins. These were aspartic, cysteine and serine-type proteases.…”

Section: Heterologous Protein Production Using Pichia Pastoris Expresmentioning

confidence: 99%

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Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,096

633

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The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

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Section: Heterologous Protein Production Using Pichia Pastoris Expresmentioning

confidence: 99%

“…The use of yeast extract and casamino acids increased protein secretion and accumulation [104], and L-arginine and EDTA increased accumulation of a single-chain antibody (scFv) three-to five-fold, although no expression levels were mentioned specifically [98].…”

Section: Nutrient Considerationsmentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,096

633

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“…For both mutants (rTLXI [H10A] and rTLXI [H22A] ) small scale expression (1.0 ml) was performed according to Shi and co-workers [20]. Large-scale expression and purification of recombinant TLXI (rTLXI) and rTLXI [H22A] were performed according to [12].…”

Section: Recombinant Expression and Purificationmentioning

confidence: 99%

His22 of TLXI plays a critical role in the inhibition of glycoside hydrolase family 11 xylanases

Rombouts

Fierens

Vandermarliere

et al. 2008

Journal of Enzyme Inhibition and Medicinal Chemistry

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Recently, a novel wheat thaumatin-like protein, TLXI, which inhibits microbial glycoside hydrolase family (GH) 11 xylanases has been identified. It is the first xylanase inhibitor that exerts its inhibition in a non-competitive way. In the present study we gained insight into the interaction between TLXI and xylanases via combined molecular modeling and mutagenic approaches. More specifically, site-specific mutation of His22, situated on a loop which distinguishes TLXI from other, non-inhibiting, thaumatin-like proteins, and subsequent expression of the mutant in Pichia pastoris resulted in a protein lacking inhibition capacity. The mutant protein was unable to form a complex with GH11 xylanases. Based on these findings, the interaction of TLXI with GH11 xylanases is discussed.

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“…Although a 28 kDa protein was detected in the culture supernatant, we observed the presence of two bands with molecular weights of ~28 and 30 kDa in polyacrylamide gels after purification with nickel affinity chromatography. Some studies also reported the expression of two bands of scFv in P. pastoris; however, they attributed this to degradation 22,23 or to incomplete cleavage of the signal sequence. 24 Other studies indicate that the additional bands detected may be due to the glycosylation of recombinant proteins with the addition of mannose residues that increase the recombinant protein molecular weight.…”

Section: Resultsmentioning

confidence: 99%